Gene expression systems in corynebacteria.

PubMed

Srivastava, Preeti; Deb, J K

2005-04-01

Corynebacterium belongs to a group of gram-positive bacteria having moderate to high G+C content, the other members being Mycobacterium, Nocardia, and Rhodococcus. Considerable information is now available on the plasmids, gene regulatory elements, and gene expression in corynebacteria, especially in soil corynebacteria such as Corynebacterium glutamicum. These bacteria are non-pathogenic and, unlike Bacillus and Streptomyces, are low in proteolytic activity and thus have the potential of becoming attractive systems for expression of heterologous proteins. This review discusses recent advances in our understanding of the organization of various regulatory elements, such as promoters, transcription terminators, and development of vectors for cloning and gene expression.

Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

PubMed

Linares, Daniel M; Alvarez-Sieiro, Patricia; del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Martin, Ma Cruz; Fernandez, Maria; Alvarez, Miguel A

2015-12-30

Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes. This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten. The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

Proposal of Self-Learning and Recognition System of Facial Expression

NASA Astrophysics Data System (ADS)

Ogawa, Yukihiro; Kato, Kunihito; Yamamoto, Kazuhiko

We describe realization of more complicated function by using the information acquired from some equipped unripe functions. The self-learning and recognition system of the human facial expression, which achieved under the natural relation between human and robot, are proposed. The robot with this system can understand human facial expressions and behave according to their facial expressions after the completion of learning process. The system modelled after the process that a baby learns his/her parents’ facial expressions. Equipping the robot with a camera the system can get face images and equipping the CdS sensors on the robot’s head the robot can get the information of human action. Using the information of these sensors, the robot can get feature of each facial expression. After self-learning is completed, when a person changed his facial expression in front of the robot, the robot operates actions under the relevant facial expression.

Transgenic Arabidopsis Gene Expression System

NASA Technical Reports Server (NTRS)

Ferl, Robert; Paul, Anna-Lisa

2009-01-01

The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

Construction of two Lactococcus lactis expression vectors combining the Gateway and the NIsin Controlled Expression systems.

PubMed

Douillard, François P; Mahony, Jennifer; Campanacci, Valérie; Cambillau, Christian; van Sinderen, Douwe

2011-09-01

Over the last 10 years, the NIsin Controlled Expression (NICE) system has been extensively used in the food-grade bacterium Lactococcus lactis subsp. cremoris to produce homologous and heterologous proteins for academic and biotechnological purposes. Although various L. lactis molecular tools have been developed, no expression vectors harboring the popular Gateway recombination system are currently available for this widely used cloning host. In this study, we constructed two expression vectors that combine the NICE and the Gateway recombination systems and we tested their applicability by recombining and over-expressing genes encoding structural proteins of lactococcal phages Tuc2009 and TP901-1. Over-expressed phage proteins were analyzed by immunoblotting and purified by His-tag affinity chromatography with protein productions yielding 2.8-3.7 mg/l of culture. This therefore is the first description of L. lactis NICE expression vectors which integrate the Gateway cloning technology and which are suitable for the production of sufficient amounts of proteins to facilitate subsequent structural and functional analyses. Copyright © 2011 Elsevier Inc. All rights reserved.

Facial expression system on video using widrow hoff

NASA Astrophysics Data System (ADS)

Jannah, M.; Zarlis, M.; Mawengkang, H.

2018-03-01

Facial expressions recognition is one of interesting research. This research contains human feeling to computer application Such as the interaction between human and computer, data compression, facial animation and facial detection from the video. The purpose of this research is to create facial expression system that captures image from the video camera. The system in this research uses Widrow-Hoff learning method in training and testing image with Adaptive Linear Neuron (ADALINE) approach. The system performance is evaluated by two parameters, detection rate and false positive rate. The system accuracy depends on good technique and face position that trained and tested.

21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cardiac allograft gene expression profiling test... Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system. (a) Identification. A cardiac allograft gene expression profiling test system is a device that measures the...

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.

Cell-Free Optogenetic Gene Expression System.

PubMed

Jayaraman, Premkumar; Yeoh, Jing Wui; Jayaraman, Sudhaghar; Teh, Ai Ying; Zhang, Jingyun; Poh, Chueh Loo

2018-04-20

Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.

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

Effects of prebiotics on immune system and cytokine expression.

PubMed

Shokryazdan, Parisa; Faseleh Jahromi, Mohammad; Navidshad, Bahman; Liang, Juan Boo

2017-02-01

Nowadays, use of prebiotics as feed and food additives has received increasing interest because of the beneficial effects of prebiotics on the health of animals and humans. One of the beneficial effects of prebiotics is stimulation of immune system, which can be direct or indirect through increasing population of beneficial microbes or probiotics, especially lactic acid bacteria and bifidobacteria, in the gut. An important mechanism of action of probiotics and prebiotics, by which they can affect the immune system, is changing the expression of cytokines. The present review tried to summarize the findings of studies that investigated the effects of prebiotics on immune system with focusing on their effects on cytokine expression. Generally, most of reviewed studies indicated beneficial effects for prebiotics in terms of improving immune system, by increasing the expression of anti-inflammatory cytokines, while reducing the expressions of proinflammatory cytokines. However, most of studies mainly considered the indirect effects of prebiotics on the immune system (through changing the composition and population of gut microbiota), and their direct effects still need to be further studied using prebiotics with different degree of polymerization in different hosts.

Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes.

PubMed

Bjerga, Gro Elin Kjæreng; Lale, Rahmi; Williamson, Adele Kim

2016-01-01

Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.

Efficient expression systems for cysteine proteases of malaria parasites

PubMed Central

Sarduy, Emir Salas; de los A. Chávez Planes, María

2013-01-01

Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes. PMID:23018863

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

PubMed

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

2017-08-01

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

Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

PubMed

Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

2017-09-15

Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.

PubMed

Guan, Chengran; Cui, Wenjing; Cheng, Jintao; Zhou, Li; Guo, Junling; Hu, Xu; Xiao, Guoping; Zhou, Zhemin

2015-09-21

Bacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA). The promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the -10 and -35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level. The auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression

Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system.

PubMed

Yan, Kelly; Merritt, Hanne; Crawford, Kenneth; Pardee, Gwynn; Cheng, Jan Marie; Widger, Stephania; Hekmat-Nejad, Mohammad; Zaror, Isabel; Sim, Janet

2015-06-01

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies. Copyright © 2015 Elsevier Inc. All rights reserved.

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system.

PubMed

Sudomoina, Marina; Latypova, Ekaterina; Favorova, Olga O; Golemis, Erica A; Serebriiskii, Ilya G

2004-04-29

Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.

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

PubMed

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

2017-10-01

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

[Pichia pastoris as an expression system for recombinant protein production].

PubMed

Ciarkowska, Anna; Jakubowska, Anna

2013-01-01

Pichia pastoris has become increasingly popular as a host for recombinant protein production in recent years. P. pastoris is more cost effective and allows achieving higher expression levels than insect and mammalian cells. It also offers some significant advantages over E. coli expression systems, such as avoiding problems with proper protein folding. Also, P. pastoris as an eukaryotic organism can carry out posttranslational modifications of produced proteins. Additionally, P. pastoris can produce high levels of recombinant proteins in extracellular medium which simplifies protein purification. Having many advantages over other expression systems makes P. pastoris an organism of choice for industrial protein production.

Expression of monellin in a food-grade delivery system in Saccharomyces cerevisiae.

PubMed

Liu, Jun; Yan, Da-zhong; Zhao, Sheng-jun

2015-10-01

Genetically modified (GM) foods have caused much controversy. Construction of a food-grade delivery system is a desirable technique with presumptive impact on industrial applications from the perspective of bio-safety. The aim of this study was to construct a food-grade delivery system for Saccharomyces cerevisiae and to study the expression of monellin from the berries of the West African forest plant Dioscoreophyllum cumminsii in this system. A food-grade system for S. cerevisiae was constructed based on ribosomal DNA (rDNA)-mediated homologous recombination to enable high-copy-number integration of the expression cassette inserted into the rDNA locus. A copper resistance gene (CUP1) was used as the selection marker for yeast transformation. Because variants of transformants containing different copy numbers at the CUP1 locus can be readily selected after growth in the presence of elevated copper levels, we suggest that this system would prove useful in the generation of tandemly iterated gene clusters. Using this food-grade system, a single-chain monellin gene was heterologously expressed. The yield of monellin reached a maximum of 675 mg L(-1) . This system harbors exclusively S. cerevisiae DNA with no antibiotic resistance genes, and it should therefore be appropriate for safe use in the food industry. Monellin was shown to be expressed in this food-grade delivery system. To our knowledge, this is the first report so far on expression of monellin in a food-grade expression system in S. cerevisiae. © 2014 Society of Chemical Industry.

Using heterologous expression systems to characterize potassium and sodium transport activities.

PubMed

Rodríguez, Alonso; Benito, Begoña; Cagnac, Olivier

2012-01-01

The expression of plant transporters in simple well-characterized cell systems is an irreplaceable technique for gaining insights into the kinetic and energetic features of plant transporters. Among all the available expression systems, yeast cells offer the highest simplicity and have the capacity to mimic the in vivo properties of plant transporters. Here, we describe the use of yeast mutants to express K(+) and Na(+) plant transporters and discuss some experimental problems that can produce misleading results.

Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILES.

PubMed

Baril, Patrick; Pichon, Chantal

2016-01-01

MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum.

PubMed

Heiss, Silvia; Hörmann, Angelika; Tauer, Christopher; Sonnleitner, Margot; Egger, Esther; Grabherr, Reingard; Heinl, Stefan

2016-03-10

Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system. Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a

Orbital Express fluid transfer demonstration system

NASA Astrophysics Data System (ADS)

Rotenberger, Scott; SooHoo, David; Abraham, Gabriel

2008-04-01

Propellant resupply of orbiting spacecraft is no longer in the realm of high risk development. The recently concluded Orbital Express (OE) mission included a fluid transfer demonstration that operated the hardware and control logic in space, bringing the Technology Readiness Level to a solid TRL 7 (demonstration of a system prototype in an operational environment). Orbital Express (funded by the Defense Advanced Research Projects Agency, DARPA) was launched aboard an Atlas-V rocket on March 9th, 2007. The mission had the objective of demonstrating technologies needed for routine servicing of spacecraft, namely autonomous rendezvous and docking, propellant resupply, and orbital replacement unit transfer. The demonstration system used two spacecraft. A servicing vehicle (ASTRO) performed multiple dockings with the client (NextSat) spacecraft, and performed a variety of propellant transfers in addition to exchanges of a battery and computer. The fluid transfer and propulsion system onboard ASTRO, in addition to providing the six degree-of-freedom (6 DOF) thruster system for rendezvous and docking, demonstrated autonomous transfer of monopropellant hydrazine to or from the NextSat spacecraft 15 times while on orbit. The fluid transfer system aboard the NextSat vehicle was designed to simulate a variety of client systems, including both blowdown pressurization and pressure regulated propulsion systems. The fluid transfer demonstrations started with a low level of autonomy, where ground controllers were allowed to review the status of the demonstration at numerous points before authorizing the next steps to be performed. The final transfers were performed at a full autonomy level where the ground authorized the start of a transfer sequence and then monitored data as the transfer proceeded. The major steps of a fluid transfer included the following: mate of the coupling, leak check of the coupling, venting of the coupling, priming of the coupling, fluid transfer, gauging

High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system.

PubMed

Martin, G A; Kawaguchi, R; Lam, Y; DeGiovanni, A; Fukushima, M; Mutter, W

2001-10-01

The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

Hierarchical Recognition Scheme for Human Facial Expression Recognition Systems

PubMed Central

Siddiqi, Muhammad Hameed; Lee, Sungyoung; Lee, Young-Koo; Khan, Adil Mehmood; Truc, Phan Tran Ho

2013-01-01

Over the last decade, human facial expressions recognition (FER) has emerged as an important research area. Several factors make FER a challenging research problem. These include varying light conditions in training and test images; need for automatic and accurate face detection before feature extraction; and high similarity among different expressions that makes it difficult to distinguish these expressions with a high accuracy. This work implements a hierarchical linear discriminant analysis-based facial expressions recognition (HL-FER) system to tackle these problems. Unlike the previous systems, the HL-FER uses a pre-processing step to eliminate light effects, incorporates a new automatic face detection scheme, employs methods to extract both global and local features, and utilizes a HL-FER to overcome the problem of high similarity among different expressions. Unlike most of the previous works that were evaluated using a single dataset, the performance of the HL-FER is assessed using three publicly available datasets under three different experimental settings: n-fold cross validation based on subjects for each dataset separately; n-fold cross validation rule based on datasets; and, finally, a last set of experiments to assess the effectiveness of each module of the HL-FER separately. Weighted average recognition accuracy of 98.7% across three different datasets, using three classifiers, indicates the success of employing the HL-FER for human FER. PMID:24316568

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

PubMed Central

Legastelois, Isabelle; Buffin, Sophie; Peubez, Isabelle; Mignon, Charlotte; Sodoyer, Régis; Werle, Bettina

2017-01-01

ABSTRACT The increasing demand for recombinant vaccine antigens or immunotherapeutic molecules calls into question the universality of current protein expression systems. Vaccine production can require relatively low amounts of expressed materials, but represents an extremely diverse category consisting of different target antigens with marked structural differences. In contrast, monoclonal antibodies, by definition share key molecular characteristics and require a production system capable of very large outputs, which drives the quest for highly efficient and cost-effective systems. In discussing expression systems, the primary assumption is that a universal production platform for vaccines and immunotherapeutics will unlikely exist. This review provides an overview of the evolution of traditional expression systems, including mammalian cells, yeast and E.coli, but also alternative systems such as other bacteria than E. coli, transgenic animals, insect cells, plants and microalgae, Tetrahymena thermophila, Leishmania tarentolae, filamentous fungi, cell free systems, and the incorporation of non-natural amino acids. PMID:27905833

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba

2014-01-01

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger.

PubMed

Geib, Elena; Brock, Matthias

2017-01-01

Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires gene activation or heterologous expression. For heterologous gene expression, we previously developed an expression platform in Aspergillus niger that is based on the transcriptional regulator TerR and its target promoter P terA . In this study, we extended this system by regulating expression of terR  by the doxycycline inducible Tet-on system. Reporter genes cloned under the control of the target promoter P terA remained silent in the absence of doxycycline, but were strongly expressed when doxycycline was added. Reporter quantification revealed that the coupled system results in about five times higher expression rates compared to gene expression under direct control of the Tet-on system. As production of secondary metabolites generally requires the expression of several biosynthetic genes, the suitability of the self-cleaving viral peptide sequence P2A was tested in this optimised expression system. P2A allowed polycistronic expression of genes required for Asp-melanin formation in combination with the gene coding for the red fluorescent protein tdTomato. Gene expression and Asp-melanin formation was prevented in the absence of doxycycline and strongly induced by addition of doxycycline. Fluorescence studies confirmed the correct subcellular localisation of the respective enzymes. This tightly regulated but strongly inducible expression system enables high level production of secondary metabolites most likely even those with toxic potential. Furthermore, this system is compatible with polycistronic gene expression and, thus, suitable for the discovery of novel natural products.

Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

PubMed

Brödel, A K; Raymond, J A; Duman, J G; Bier, F F; Kubick, S

2013-02-10

Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system.

PubMed

da Silva Junior, Haroldo Cid; da Silva E Mouta Junior, Sérgio; de Mendonça, Marcos César Lima; de Souza Pereira, Mirian Claudia; da Rocha Nogueira, Alanderson; de Azevedo, Maria Luiza Borges; Leite, José Paulo Gagliardi; de Moraes, Márcia Terezinha Baroni

2012-09-01

The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.

Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

PubMed

Steinmetz, Eric J; Auldridge, Michele E

2017-11-01

The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

The Facial Expression Coding System (FACES): Development, Validation, and Utility

ERIC Educational Resources Information Center

Kring, Ann M.; Sloan, Denise M.

2007-01-01

This article presents information on the development and validation of the Facial Expression Coding System (FACES; A. M. Kring & D. Sloan, 1991). Grounded in a dimensional model of emotion, FACES provides information on the valence (positive, negative) of facial expressive behavior. In 5 studies, reliability and validity data from 13 diverse…

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

PubMed

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

2015-01-16

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

Bacterial cell-free expression technology to in vitro systems engineering and optimization.

PubMed

Caschera, Filippo

2017-06-01

Cell-free expression system is a technology for the synthesis of proteins in vitro . The system is a platform for several bioengineering projects, e.g. cell-free metabolic engineering, evolutionary design of experiments, and synthetic minimal cell construction. Bacterial cell-free protein synthesis system (CFPS) is a robust tool for synthetic biology. The bacteria lysate, the DNA, and the energy module, which are the three optimized sub-systems for in vitro protein synthesis, compose the integrated system. Currently, an optimized E. coli cell-free expression system can produce up to ∼2.3 mg/mL of a fluorescent reporter protein. Herein, I will describe the features of ATP-regeneration systems for in vitro protein synthesis, and I will present a machine-learning experiment for optimizing the protein yield of E. coli cell-free protein synthesis systems. Moreover, I will introduce experiments on the synthesis of a minimal cell using liposomes as dynamic containers, and E. coli cell-free expression system as biochemical platform for metabolism and gene expression. CFPS can be further integrated with other technologies for novel applications in environmental, medical and material science.

21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system. (a...

21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system. (a...

21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system. (a...

21 CFR 862.1163 - Cardiac allograft gene expression profiling test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1163 Cardiac allograft gene expression profiling test system. (a...

The growing impact of lyophilized cell-free protein expression systems

PubMed Central

Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

2017-01-01

ABSTRACT Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis. PMID:27791452

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

PubMed

Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

2018-10-01

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Highly repressible expression system for cloning genes that specify potentially toxic proteins.

PubMed Central

O'Connor, C D; Timmis, K N

1987-01-01

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational. Images PMID:2443481

Dynamic Visualization of Co-expression in Systems Genetics Data

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

New, Joshua Ryan; Huang, Jian; Chesler, Elissa J

2008-01-01

Biologists hope to address grand scientific challenges by exploring the abundance of data made available through modern microarray technology and other high-throughput techniques. The impact of this data, however, is limited unless researchers can effectively assimilate such complex information and integrate it into their daily research; interactive visualization tools are called for to support the effort. Specifically, typical studies of gene co-expression require novel visualization tools that enable the dynamic formulation and fine-tuning of hypotheses to aid the process of evaluating sensitivity of key parameters. These tools should allow biologists to develop an intuitive understanding of the structure of biologicalmore » networks and discover genes which reside in critical positions in networks and pathways. By using a graph as a universal data representation of correlation in gene expression data, our novel visualization tool employs several techniques that when used in an integrated manner provide innovative analytical capabilities. Our tool for interacting with gene co-expression data integrates techniques such as: graph layout, qualitative subgraph extraction through a novel 2D user interface, quantitative subgraph extraction using graph-theoretic algorithms or by querying an optimized b-tree, dynamic level-of-detail graph abstraction, and template-based fuzzy classification using neural networks. We demonstrate our system using a real-world workflow from a large-scale, systems genetics study of mammalian gene co-expression.« less

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

PubMed

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

2018-05-18

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

Systemic bioinformatics analysis of skeletal muscle gene expression profiles of sepsis

PubMed Central

Yang, Fang; Wang, Yumei

2018-01-01

Sepsis is a type of systemic inflammatory response syndrome with high morbidity and mortality. Skeletal muscle dysfunction is one of the major complications of sepsis that may also influence the outcome of sepsis. The aim of the present study was to explore and identify potential mechanisms and therapeutic targets of sepsis. Systemic bioinformatics analysis of skeletal muscle gene expression profiles from the Gene Expression Omnibus was performed. Differentially expressed genes (DEGs) in samples from patients with sepsis and control samples were screened out using the limma package. Differential co-expression and coregulation (DCE and DCR, respectively) analysis was performed based on the Differential Co-expression Analysis package to identify differences in gene co-expression and coregulation patterns between the control and sepsis groups. Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways of DEGs were identified using the Database for Annotation, Visualization and Integrated Discovery, and inflammatory, cancer and skeletal muscle development-associated biological processes and pathways were identified. DCE and DCR analysis revealed several potential therapeutic targets for sepsis, including genes and transcription factors. The results of the present study may provide a basis for the development of novel therapeutic targets and treatment methods for sepsis. PMID:29805480

Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

DOE PAGES

Liang, Yan; Richardson, Sarah; Yan, Jingwei; ...

2017-01-17

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

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

Liang, Yan; Richardson, Sarah; Yan, Jingwei

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

Orbital express capture system: concept to reality

NASA Astrophysics Data System (ADS)

Stamm, Shane; Motaghedi, Pejmun

2004-08-01

The development of autonomous servicing of on-orbit spacecraft has been a sought after objective for many years. A critical component of on-orbit servicing involves the ability to successfully capture, institute mate, and perform electrical and fluid transfers autonomously. As part of a Small Business Innovation Research (SBIR) grant, Starsys Research Corporation (SRC) began developing such a system. Phase I of the grant started in 1999, with initial work focusing on simultaneously defining the parameters associated with successful docking while designing to those parameters. Despite the challenge of working without specific requirements, SRC completed development of a prototype design in 2000. Throughout the following year, testing was conducted on the prototype to characterize its performance. Having successfully completed work on the prototype, SRC began a Phase II SBIR effort in mid-2001. The focus of the second phase was a commercialization effort designed to augment the prototype model into a more flight-like design. The technical requirements, however, still needed clear definition for the design to progress. The advent of the Orbital Express (OE) program provided much of that definition. While still in the proposal stages of the OE program, SRC began tailoring prototype redesign efforts to the OE program requirements. A primary challenge involved striking a balance between addressing the technical requirements of OE while designing within the scope of the SBIR. Upon award of the OE contract, the Phase II SBIR design has been fully developed. This new design, designated the Mechanical Docking System (MDS), successfully incorporated many of the requirements of the OE program. SRC is now completing dynamic testing on the MDS hardware, with a parallel effort of developing a flight design for OE. As testing on the MDS progresses, the design path that was once common to both SBIR effort and the OE program begins to diverge. The MDS will complete the scope of the

Differential expression of neuroligin genes in the nervous system of zebrafish.

PubMed

Davey, Crystal; Tallafuss, Alexandra; Washbourne, Philip

2010-02-01

The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance.

Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

PubMed

Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

2013-01-01

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

The heterologous expression strategies of antimicrobial peptides in microbial systems.

PubMed

Deng, Ting; Ge, Haoran; He, Huahua; Liu, Yao; Zhai, Chao; Feng, Liang; Yi, Li

2017-12-01

Antimicrobial peptides (AMPs) consist of molecules acting on the defense systems of numerous organisms toward tumor and multiple pathogens, such as bacteria, fungi, viruses, and parasites. Compared to traditional antibiotics, AMPs are more stable and have lower propensity for developing resistance through functioning in the innate immune system, thus having important applications in the fields of medicine, food and so on. However, despite of their high economic values, the low yield and the cumbersome extraction process in AMPs production are problems that limit their industrial application and scientific research. To conquer these obstacles, optimized heterologous expression technologies were developed that could provide effective ways to increase the yield of AMPs. In this review, the research progress on heterologous expression of AMPs using Escherichia coli, Bacillus subtilis, Pichia pastoris and Saccharomyces cerevisiae as host cells was mainly summarized, which might guide the expression strategies of AMPs in these cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803.

PubMed

Abe, Koichi; Miyake, Kotone; Nakamura, Mayumi; Kojima, Katsuhiro; Ferri, Stefano; Ikebukuro, Kazunori; Sode, Koji

2014-03-01

In order to construct a green-light-regulated gene expression system for cyanobacteria, we characterized a green-light sensing system derived from Synechocystis sp. PCC6803, consisting of the green-light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (PcpcG 2 ). CcaS and CcaR act as a genetic controller and activate gene expression from PcpcG 2 with green-light illumination. The green-light induction level of the native PcpcG 2 was investigated using GFPuv as a reporter gene inserted in a broad-host-range vector. A clear induction of protein expression from native PcpcG 2 under green-light illumination was observed; however, the expression level was very low compared with Ptrc , which was reported to act as a constitutive promoter in cyanobacteria. Therefore, a Shine-Dalgarno-like sequence derived from the cpcB gene was inserted in the 5' untranslated region of the cpcG2 gene, and the expression level of CcaR was increased. Thus, constructed engineered green-light sensing system resulted in about 40-fold higher protein expression than with the wild-type promoter with a high ON/OFF ratio under green-light illumination. The engineered green-light gene expression system would be a useful genetic tool for controlling gene expression in the emergent cyanobacterial bioprocesses. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

PubMed

Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

2017-01-01

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

Candidate innate immune system gene expression in the ecological model Daphnia

PubMed Central

Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E.; Little, Tom J.

2011-01-01

The last ten years have witnessed increasing interest in host–pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host–pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia–pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia–Pasteuria system will need to balance a candidate gene approach with more

Candidate innate immune system gene expression in the ecological model Daphnia.

PubMed

Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E; Little, Tom J

2011-10-01

The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive

Analytical expressions for the nonlinear interference in dispersion managed transmission coherent optical systems

NASA Astrophysics Data System (ADS)

Qiao, Yaojun; Li, Ming; Yang, Qiuhong; Xu, Yanfei; Ji, Yuefeng

2015-01-01

Closed-form expressions of nonlinear interference of dense wavelength-division-multiplexed (WDM) systems with dispersion managed transmission (DMT) are derived. We carry out a simulative validation by addressing an ample and significant set of the Nyquist-WDM systems based on polarization multiplexed quadrature phase-shift keying (PM-QPSK) subcarriers at a baud rate of 32 Gbaud per channel. Simulation results show the simple closed-form analytical expressions can provide an effective tool for the quick and accurate prediction of system performance in DMT coherent optical systems.

Identification and expression of the protein ubiquitination system in Giardia intestinalis.

PubMed

Gallego, Eva; Alvarado, Magda; Wasserman, Moises

2007-06-01

Giardia intestinalis is a single-cell eukaryotic microorganism, regarded as one of the earliest divergent eukaryotes and thus an attractive model to study the evolution of regulatory systems. Giardia has two different forms throughout its life cycle, cyst and trophozoite, and changes from one to the other in response to environmental signals. The two differentiation processes involve a differential gene expression as well as a quick and specific protein turnover that may be mediated by the ubiquitin/proteasome system. The aim of this work was to search for unreported components of the ubiquitination system and to experimentally demonstrate their expression in the parasite and during the two differentiation processes. We found activity of protein ubiquitination in G. intestinalis trophozoites and analyzed the transcription of the ubiquitin gene, as well as that of the activating (E1), conjugating (E2), and ligase (E3) ubiquitin enzymes during encystation and excystation. A constant ubiquitin expression persisted during the parasite's differentiation processes, whereas variation in transcription was observed in the other genes under study.

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system.

PubMed

Ruppert, Martin; Woll, Jörn; Giritch, Anatoli; Genady, Ezzat; Ma, Xueyan; Stöckigt, Joachim

2005-11-01

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

PubMed

Kao, Tim; Labonne, Tanya; Niclis, Jonathan C; Chaurasia, Ritu; Lokmic, Zerina; Qian, Elizabeth; Bruveris, Freya F; Howden, Sara E; Motazedian, Ali; Schiesser, Jacqueline V; Costa, Magdaline; Sourris, Koula; Ng, Elizabeth; Anderson, David; Giudice, Antonietta; Farlie, Peter; Cheung, Michael; Lamande, Shireen R; Penington, Anthony J; Parish, Clare L; Thomson, Lachlan H; Rafii, Arash; Elliott, David A; Elefanty, Andrew G; Stanley, Edouard G

2016-09-13

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.

PubMed

Rozov, S M; Permyakova, N V; Deineko, E V

2018-03-01

Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.

Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

PubMed

Son, Seung-Myoung; Ha, Sang-Yun; Yoo, Hae-Yong; Oh, Dongryul; Kim, Seok-Jin; Kim, Won-Seog; Ko, Young-Hyeh

2017-01-01

The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

Expression of glypican-3 is highly associated with pediatric hepatoblastoma: a systemic analysis.

PubMed

Xiong, Xiao-Li; Qin, Huan; Yan, Su-Qi; Zhou, Li-Shan; Chen, Peng; Zhao, Dong- Chi

2015-01-01

Glypican-3 (GPC3) is reported to be an oncofetal protein that is a useful diagnostic immunomarker for hepatoblastoma. However, the results are not inclusive. This study systemically investigated the association between expression of GPC3 and pediatric hepatoblastoma. Clinical studies evaluating the association were identified using a predefined search strategy. GPC3 immunohistochemistry was applied in the pathological diagnosis of hepatoblastoma using the monoclonal antibodies with formalin-fixed and paraffin-embedded specimens. Positive predictive rates for the association between expression of GPC3 and pediatric hepatoblastoma were calculated. Specimens from four clinical studies which including 134 patients with pediatric hepatoblastoma tested by GPC3 immunohistochemistry were considered eligible for inclusion. Systemic analysis showed that, in all patients, pooled positive predictive rate of the association between expression of GPC3 and pediatric hepatoblastoma was 95.5% (128/134). This systemic analysis suggests that the expression of glypican-3 is highly associated with the diagnosis of pediatric hepatoblastoma.

Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation

PubMed Central

Soto, Lilian; Ferrier, Ashley; Aravena, Octavio; Fonseca, Elianet; Berendsen, Jorge; Biere, Andrea; Bueno, Daniel; Ramos, Verónica; Aguillón, Juan Carlos; Catalán, Diego

2015-01-01

The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis. PMID:26483788

AMTEC radioisotope power system design and analysis for Pluto Express Fly-By

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

Hendricks, T.J.; Huang, C.; Sievers, R.K.

1997-12-31

The Pluto Express Fly-By program requires a Radioisotope Power System (RPS) to supply spacecraft power for various internal functions and mission instruments and experiments. AMTEC (Alkali-Metal Thermal-Electric Conversion) power conversion is the DOE-selected technology for an advanced, high-efficiency RPS to power the Pluto Express Fly-By spacecraft. An AMTEC-based RPS using the General Purpose Heat Source (GPHS) has been conceptually designed to satisfy the Pluto Express power requirements. Integrated AMTEC cell and system thermal/electrical design analyses, structural design analyses, and mass analyses were performed to define an optimum system design. Using fresh radioisotope fuel at beginning of mission, the RPS producesmore » 102 watts of power, has a mass of 8.35 kg (specific power density = 12.2 watts/kg), with a system conversion efficiency of 20.3%. Mass/power scale-up estimates have also been generated, indicating that a 150-watt version of this RPS would weigh approximately 11.3 kg. This paper presents and discusses the key features of this RPS design, the design and analysis methodology, and the numerous system and AMTEC cell tradeoff studies establishing the optimum AMTEC-based RPS.« less

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

PubMed Central

2011-01-01

Background Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies. Results The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells. Conclusion Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all

On the use of the T-REx tetracycline-inducible gene expression system in vivo.

PubMed

Dobrovolsky, Vasily N; Heflich, Robert H

2007-10-15

Components of the commercially available T-REx system were used to create two types of transgenic mice. The first contained the tetracycline-repressor transgene under the control of the CMV promoter/enhancer; the second type contained a green fluorescent protein (GFP) reporter transgene under the control of the CMV promoter/enhancer with a tetracycline repressor operator sequence. Transgene expression was unpredictable in animals containing the individual transgenes. Animals with the reporter transgene expressed GFP in only some tissues (e.g., pancreas, kidney), and one line of reporter transgenic animals developed kidney disease, presumably due to expression of the transgene. The two types of transgenic animals were crossbred to produce double-transgenic animals with the object of regulating the expression of the reporter in vivo. When a similar double-transgenic system was constructed in cultured cells, the repressor protein suppressed the transcription of the reporter transgene. The presence of the repressor in double-transgenic animals had no effect on the expression of the reporter; double transgenic animals developed the same kidney disease that was seen in singly transgenic mice with the reporter. Our results indicate that transgenes under the control of the CMV promoter in the T-REx system express somewhat unpredictably and in only a limited number of tissues, making the use of this system for the development of in vivo models problematical. Copyright 2007 Wiley Periodicals, Inc.

FPGA-accelerated algorithm for the regular expression matching system

NASA Astrophysics Data System (ADS)

Russek, P.; Wiatr, K.

2015-01-01

This article describes an algorithm to support a regular expressions matching system. The goal was to achieve an attractive performance system with low energy consumption. The basic idea of the algorithm comes from a concept of the Bloom filter. It starts from the extraction of static sub-strings for strings of regular expressions. The algorithm is devised to gain from its decomposition into parts which are intended to be executed by custom hardware and the central processing unit (CPU). The pipelined custom processor architecture is proposed and a software algorithm explained accordingly. The software part of the algorithm was coded in C and runs on a processor from the ARM family. The hardware architecture was described in VHDL and implemented in field programmable gate array (FPGA). The performance results and required resources of the above experiments are given. An example of target application for the presented solution is computer and network security systems. The idea was tested on nearly 100,000 body-based viruses from the ClamAV virus database. The solution is intended for the emerging technology of clusters of low-energy computing nodes.

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

PubMed

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

2008-09-01

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

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

PubMed Central

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

2015-01-01

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

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

PubMed

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

2018-04-18

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

A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

PubMed Central

2017-01-01

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. PMID:29084263

A tetracycline expression system in combination with Sox9 for cartilage tissue engineering.

PubMed

Yao, Yi; He, Yu; Guan, Qian; Wu, Qiong

2014-02-01

Cartilage tissue engineering using controllable transcriptional therapy together with synthetic biopolymer scaffolds shows higher potential for overcoming chondrocyte degradation and constructing artificial cartilages both in vivo and in vitro. Here, the potential regulating tetracycline expression (Tet-on) system was used to express Sox9 both in vivo and in vitro. Chondrocyte degradation was measured in vitro and overcome by Soxf9 expression. Experiments confirmed the feasibility of the combined use of Sox9 and Tet-on system in cartilage tissue engineering. Engineered poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) scaffolds were seeded with recombinant chondrocytes which were transfected with Tet-induced Sox9 expression; the scaffolds were implanted under the skin of 8-week-old rats. The experimental group was injected with Dox in the abdomen, while the control group was injected with normal saline. After 4 or 8 days of implantation in vivo, the newly formed pieces of articular chondrocytes were taken out and measured. Dox injection in vivo showed positive effect on recombinant chondrocytes, in which Sox9 expression was up-regulated by an inducible system with specific matrix proteins. The results demonstrate this controllable transcriptional therapy is a potential approach for tissue engineering. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

PubMed

Shi, Yingli; Xiang, Jianhai; Zhou, Guangzhou; Ron, Tetsuzan Benny; Tong, Hsin-I; Kang, Wen; Sun, Si; Lu, Yuanan

2016-06-01

A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.

Expression of Lectins in Heterologous Systems

PubMed Central

Martínez-Alarcón, Dania; Blanco-Labra, Alejandro

2018-01-01

Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, there is great interest in their production on a large scale. Unfortunately, conventional techniques do not provide the appropriate platform for this purpose and therefore, the heterologous production of lectins in different organisms has become the preferred method in many cases. Such systems have the advantage of providing better yields as well as more homogeneous and better-defined properties for the resultant products. However, an inappropriate choice of the expression system can cause important structural alterations that have repercussions on their biological activity since the specificity may lay in their post-translational processing, which depends largely on the producing organism. The present review aims to examine the most representative studies in the area, exposing the four most frequently used systems (bacteria, yeasts, plants and animal cells), with the intention of providing the necessary information to determine the strategy to follow in each case as well as their respective advantages and disadvantages. PMID:29466298

Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

NASA Technical Reports Server (NTRS)

Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

2003-01-01

Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

Analytical expressions for the evolution of many-body quantum systems quenched far from equilibrium

NASA Astrophysics Data System (ADS)

Santos, Lea F.; Torres-Herrera, E. Jonathan

2017-12-01

Possible strategies to describe analytically the dynamics of many-body quantum systems out of equilibrium include the use of solvable models and of full random matrices. None of the two approaches represent actual realistic systems, but they serve as references for the studies of these ones. We take the second path and obtain analytical expressions for the survival probability, density imbalance, and out-of-time-ordered correlator. Using these findings, we then propose an approximate expression that matches very well numerical results for the evolution of realistic finite quantum systems that are strongly chaotic and quenched far from equilibrium. In the case of the survival probability, the expression proposed covers all different time scales, from the moment the system is taken out of equilibrium to the moment it reaches a new equilibrium. The realistic systems considered are described by one-dimensional spin-1/2 models.

Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

PubMed Central

Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

2013-01-01

Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

Regulatory systems for hypoxia-inducible gene expression in ischemic heart disease gene therapy.

PubMed

Kim, Hyun Ah; Rhim, Taiyoun; Lee, Minhyung

2011-07-18

Ischemic heart diseases are caused by narrowed coronary arteries that decrease the blood supply to the myocardium. In the ischemic myocardium, hypoxia-responsive genes are up-regulated by hypoxia-inducible factor-1 (HIF-1). Gene therapy for ischemic heart diseases uses genes encoding angiogenic growth factors and anti-apoptotic proteins as therapeutic genes. These genes increase blood supply into the myocardium by angiogenesis and protect cardiomyocytes from cell death. However, non-specific expression of these genes in normal tissues may be harmful, since growth factors and anti-apoptotic proteins may induce tumor growth. Therefore, tight gene regulation is required to limit gene expression to ischemic tissues, to avoid unwanted side effects. For this purpose, various gene expression strategies have been developed for ischemic-specific gene expression. Transcriptional, post-transcriptional, and post-translational regulatory strategies have been developed and evaluated in ischemic heart disease animal models. The regulatory systems can limit therapeutic gene expression to ischemic tissues and increase the efficiency of gene therapy. In this review, recent progresses in ischemic-specific gene expression systems are presented, and their applications to ischemic heart diseases are discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

BRCA1 and BRCA2 expression patterns and prognostic significance in digestive system cancers.

PubMed

Wang, Gui-Hua; Zhao, Chun-Mei; Huang, Ying; Wang, Wei; Zhang, Shu; Wang, Xudong

2018-01-01

The role of BRCA1 and BRCA2 genes is mainly to maintain genome integrity in response to DNA damage through different mechanisms. Deregulation of BRCA1 and BRCA2 is associated with the development of tumor and altered sensitivity to chemotherapeutic agents. In this study, we determined protein expression of BRCA1 and BRCA2 in 4 digestive system cancers (gastric cancer, colorectal cancer, hepatocellular carcinoma, and pancreatic cancer) by immunohistochemistry on tissue microarrays. A total of 1546 samples of 4 types of cancer tissues, their matched adjacent nontumor tissues, and corresponding benign tissues were studied, respectively. Immunohistochemistry expression patterns of the 2 proteins and their correlation with patients' clinical parameters and overall survival were analyzed. The results showed that low expression of cytoplasmic BRCA1 and BRCA2 was commonly associated with advanced tumor-lymph node-metastasis stage, whereas high expression of nuclear BRCA1 was generally correlated with advanced tumor stages in these cancers. High expression of cytoplasmic BRCA1 and BRCA2 had significantly favorable overall survival in digestive system cancers; in contrast, BRCA1 nuclear expression usually predicted poor outcomes. We conclude that BRCA1 and BRCA2 could be used as clinicopathological biomarkers to evaluate the prognosis of digestive system cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression profiles of inka2 in the murine nervous system.

PubMed

Iwasaki, Yumi; Yumoto, Takahito; Sakakibara, Shin-Ichi

2015-01-01

Dynamic rearrangement of the actin cytoskeleton impacts many cellular characteristics in both the developing and adult central nervous systems (CNS), including the migration and adhesion of highly motile neural progenitor cells, axon guidance of immature neurons, and reconstruction of synaptic structures in the adult brain. Inka1, a known regulator of actin cytoskeleton reconstruction, is predominantly expressed by the neural crest cell lineage and regulates the migration and differentiation of these cells. In the present study, we identified a novel gene, designated as inka2, which is related to inka1. Inka2/fam212b is an evolutionarily conserved gene found in different vertebrate species and constitutes a novel gene family together with inka1. Northern blot analysis showed that inka2 mRNA was highly enriched in the nervous system. The spatiotemporal propagation cell profiles of those cells that expressed inka2 transcripts were compatible with those of Olig2-positive oligodendrocyte progenitor cells, which originate in the ventral ventricular zone during embryogenesis. Intense expression of inka2 was also noted in the proliferative neuronal progenitors in the developing cerebellum. On the other hand, immature newborn neurons in the embryonic brain showed no expression of inka2, except for the cells residing in the marginal zone of the embryonic telencephalon, which is known to contain transient cells including the non-subplate pioneer neurons and Cajal-Retzius cells. As brain development proceeds during the postnatal stage, inka2 expression emerged in some populations of immature neurons, including the neocortical pyramidal neurons, hippocampal pyramidal neurons, and granule cells migrating in the cerebellar cortex. In the adult brain, the expression of inka2 was interestingly confined in terminally differentiated neurons in the restricted forebrain regions. Taken together, as a novel regulator of actin cytoskeletons in the CNS, inka2 may be involved in multiple

A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

DOE PAGES

Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; ...

2015-03-18

One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated amore » native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose

Control of gene expression by CRISPR-Cas systems

PubMed Central

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems. PMID:24273648

The multiBac protein complex production platform at the EMBL.

PubMed

Berger, Imre; Garzoni, Frederic; Chaillet, Maxime; Haffke, Matthias; Gupta, Kapil; Aubert, Alice

2013-07-11

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.(1,2) Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.(3) BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.(4) A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.(5-8) The platform is installed in an open-access mode at EMBL Grenoble and has supported many

Insect cells-baculovirus system for the production of difficult to express proteins.

PubMed

Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

2015-01-01

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

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.

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

Deregulated HOXB7 expression predicts poor prognosis of patients with malignancies of digestive system.

PubMed

Liu, Fang-Teng; Chen, Han-Min; Xiong, Ying; Zhu, Zheng-Ming

2017-07-26

Numerous studies have investigated the relationship between deregulated HOXB7 expression with the clinical outcome in patients with digestive stem cancers, HOXB7 has showed negative impacts but with varying levels. We aimed to comprehensively evaluate the prediction and prognostic value of HOXB7 in digestive stem cancers. Electronic databases updated to December 1, 2016 were retrieved to collect relevant eligible studies to quantitatively explore the potential roles of HOXB7 as a prognostic indicator in digestive system cancers. A total of 9 studies (n = 1298 patients) was included in this synthetical meta-analysis. The pooled hazard ratios suggested that high expression of HOXB7 protein was associated with poor prognosis of OS in patients with digestive system cancers (HR = 1.97, 95% CI: 1.65-2.28, p= 0.000), and HOXB7 protein could act as an independent prognostic factor for predicting OS of patients with digestive system cancers (HR: 2.02, 95% CI: 1.69-2.36, p = 0.000). Statistical significance was also observed in subgroup meta-analysis based on the cancer type, histology type, country, sample size and publication date. Furthermore, we examined the correlations between HOXB7 protein and clinicopathological features. It showed that altered expression of HOXB7 protein was correlated with tumor invasion (p = 0.000), lymph node status (p = 0.000), distant metastasis (p = 0.001) and TNM stage (p = 0.000). However, the expression of HOXB7 protein was not associated with age (p = 0.64), gender (p = 0.40) or levels of differentiation (p = 0.19). High expression of HOXB7 protein was associated with poor prognosis of patients with digestive system cancers, as well as clinicopathologic characteristics, including the tumor invasion, lymph node status, distant metastasis and TNM stage. The expression of HOXB7 protein was not associated with age, gender or levels of differentiation. HOXB7 protein expression level in tumor tissue might serve as a novel prognostic marker for

Development and characterization of a eukaryotic expression system for human type II procollagen.

PubMed

Wieczorek, Andrew; Rezaei, Naghmeh; Chan, Clara K; Xu, Chuan; Panwar, Preety; Brömme, Dieter; Merschrod S, Erika F; Forde, Nancy R

2015-12-15

Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Dynamics of Immune System Gene Expression upon Bacterial Challenge and Wounding in a Social Insect (Bombus terrestris)

PubMed Central

Erler, Silvio; Popp, Mario; Lattorff, H. Michael G.

2011-01-01

The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT). There is a lack of immune genes in social insects (e.g. honeybees) when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals). The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge. Antimicrobial peptides (AMP) (abaecin, defensin 1, hymenoptaecin) were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish) and JNK pathway (basket). Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment. Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the

Expression and functional characterisation of System L amino acid transporters in the human term placenta.

PubMed

Gaccioli, Francesca; Aye, Irving L M H; Roos, Sara; Lager, Susanne; Ramirez, Vanessa I; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

2015-06-09

System L transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) mediate the uptake of large, neutral amino acids in the human placenta. Many System L substrates are essential amino acids, thus representing crucial nutrients for the growing fetus. Both LAT isoforms are expressed in the human placenta, but the relative contribution of LAT1 and LAT2 to placental System L transport and their subcellular localisation are not well established. Moreover, the influence of maternal body mass index (BMI) on placental System L amino acid transport is poorly understood. Therefore the aims of this study were to determine: i) the relative contribution of the LAT isoforms to System L transport activity in primary human trophoblast (PHT) cells isolated from term placenta; ii) the subcellular localisation of LAT transporters in human placenta; and iii) placental expression and activity of System L transporters in response to maternal overweight/obesity. System L mediated leucine uptake was measured in PHT cells after treatment with si-RNA targeting LAT1 and/or LAT2. The localisation of LAT isoforms was studied in isolated microvillous plasma membranes (MVM) and basal membranes (BM) by Western blot analysis. Results were confirmed by immunohistochemistry in sections of human term placenta. Expression and activity System L transporters was measured in isolated MVM from women with varying pre-pregnancy BMI. Both LAT1 and LAT2 isoforms contribute to System L transport activity in primary trophoblast cells from human term placenta. LAT1 and LAT2 transporters are highly expressed in the MVM of the syncytiotrophoblast layer at term. LAT2 is also localised in the basal membrane and in endothelial cells lining the fetal capillaries. Measurements in isolated MVM vesicles indicate that System L transporter expression and activity is not influenced by maternal BMI. LAT1 and LAT2 are present and functional in the syncytiotrophoblast MVM, whereas LAT2 is also expressed in the BM and in the fetal capillary

The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

PubMed Central

Mohajeri, Abbas; Sanaei, Sarvin; Kiafar, Farhad; Fattahi, Amir; Khalili, Majid; Zarghami, Nosratollah

2017-01-01

Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es) is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs) is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli) expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A) of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin. PMID:28507934

Localization of PPARdelta in murine central nervous system: expression in oligodendrocytes and neurons.

PubMed

Woods, John W; Tanen, Michael; Figueroa, David J; Biswas, Chhabi; Zycband, Emanuel; Moller, David E; Austin, Christopher P; Berger, Joel P

2003-06-13

The peroxisome proliferator-activated receptors (PPARs), PPARdelta, PPARgamma and PPARalpha, comprise a subclass of the supergene family of nuclear receptors. As such they are ligand-regulated transcription factors whose major effects are mediated by altering expression of target genes. PPARdelta has been shown to be ubiquitously expressed in mammals. However, its primary biological role(s) has yet to be defined. Several recent studies have demonstrated that PPARdelta is the most highly expressed PPAR isoform in the central nervous system, but ambiguity still exists as to the specific brain sub-regions and cells in which it is expressed. Here, utilizing novel, isoform-selective PPARdelta riboprobes and an anti-peptide antibody, we performed a series of in situ hybridization and immunolocalization studies to determine the distribution of PPARdelta in the central nervous system (CNS) of mice. We found that PPARdelta mRNA and protein is expressed throughout the brain, with particularly high levels in the entorhinal cortex, hypothalamus and hippocampus, and lower levels in the corpus callosum and caudate putamen. At the cellular level, PPARdelta mRNA and protein were found to be expressed in oligodendrocytes and neurons but not astrocytes. Such results suggest a role for PPARdelta in both myelination and neuronal functioning within the CNS.

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.

What is adapted in face adaptation? The neural representations of expression in the human visual system.

PubMed

Fox, Christopher J; Barton, Jason J S

2007-01-05

The neural representation of facial expression within the human visual system is not well defined. Using an adaptation paradigm, we examined aftereffects on expression perception produced by various stimuli. Adapting to a face, which was used to create morphs between two expressions, substantially biased expression perception within the morphed faces away from the adapting expression. This adaptation was not based on low-level image properties, as a different image of the same person displaying that expression produced equally robust aftereffects. Smaller but significant aftereffects were generated by images of different individuals, irrespective of gender. Non-face visual, auditory, or verbal representations of emotion did not generate significant aftereffects. These results suggest that adaptation affects at least two neural representations of expression: one specific to the individual (not the image), and one that represents expression across different facial identities. The identity-independent aftereffect suggests the existence of a 'visual semantic' for facial expression in the human visual system.

Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

PubMed Central

Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G.

2013-01-01

The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed. PMID:24294221

Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system.

PubMed

Rabert, Claudia; Weinacker, Daniel; Pessoa, Adalberto; Farías, Jorge G

2013-01-01

The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process; the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.

An orthogonal system for heterologous expression of actinobacterial lasso peptides in Streptomyces hosts.

PubMed

Mevaere, Jimmy; Goulard, Christophe; Schneider, Olha; Sekurova, Olga N; Ma, Haiyan; Zirah, Séverine; Afonso, Carlos; Rebuffat, Sylvie; Zotchev, Sergey B; Li, Yanyan

2018-05-29

Lasso peptides are ribosomally synthesized and post-translationally modified peptides produced by bacteria. They are characterized by an unusual lariat-knot structure. Targeted genome scanning revealed a wide diversity of lasso peptides encoded in actinobacterial genomes, but cloning and heterologous expression of these clusters turned out to be problematic. To circumvent this, we developed an orthogonal expression system for heterologous production of actinobacterial lasso peptides in Streptomyces hosts based on a newly-identified regulatory circuit from Actinoalloteichus fjordicus. Six lasso peptide gene clusters, mainly originating from marine Actinobacteria, were chosen for proof-of-concept studies. By varying the Streptomyces expression hosts and a small set of culture conditions, three new lasso peptides were successfully produced and characterized by tandem MS. The newly developed expression system thus sets the stage to uncover and bioengineer the chemo-diversity of actinobacterial lasso peptides. Moreover, our data provide some considerations for future bioprospecting efforts for such peptides.

Modular and coordinated expression of immune system regulatory and signaling components in the developing and adult nervous system.

PubMed

Monzón-Sandoval, Jimena; Castillo-Morales, Atahualpa; Crampton, Sean; McKelvey, Laura; Nolan, Aoife; O'Keeffe, Gerard; Gutierrez, Humberto

2015-01-01

During development, the nervous system (NS) is assembled and sculpted through a concerted series of neurodevelopmental events orchestrated by a complex genetic programme. While neural-specific gene expression plays a critical part in this process, in recent years, a number of immune-related signaling and regulatory components have also been shown to play key physiological roles in the developing and adult NS. While the involvement of individual immune-related signaling components in neural functions may reflect their ubiquitous character, it may also reflect a much wider, as yet undescribed, genetic network of immune-related molecules acting as an intrinsic component of the neural-specific regulatory machinery that ultimately shapes the NS. In order to gain insights into the scale and wider functional organization of immune-related genetic networks in the NS, we examined the large scale pattern of expression of these genes in the brain. Our results show a highly significant correlated expression and transcriptional clustering among immune-related genes in the developing and adult brain, and this correlation was the highest in the brain when compared to muscle, liver, kidney and endothelial cells. We experimentally tested the regulatory clustering of immune system (IS) genes by using microarray expression profiling in cultures of dissociated neurons stimulated with the pro-inflammatory cytokine TNF-alpha, and found a highly significant enrichment of immune system-related genes among the resulting differentially expressed genes. Our findings strongly suggest a coherent recruitment of entire immune-related genetic regulatory modules by the neural-specific genetic programme that shapes the NS.

Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems

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

Conover, David R.; Crawford, Aladsair J.; Fuller, Jason C.

The Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems (PNNL-22010) was first issued in November 2012 as a first step toward providing a foundational basis for developing an initial standard for the uniform measurement and expression of energy storage system (ESS) performance. Based on experiences with the application and use of that document, and to include additional ESS applications and associated duty cycles, test procedures and performance metrics, a first revision of the November 2012 Protocol was issued in June 2014 (PNNL 22010 Rev. 1). As an update of the 2014 revision 1 to the Protocol,more » this document (the March 2016 revision 2 to the Protocol) is intended to supersede the June 2014 revision 1 to the Protocol and provide a more user-friendly yet more robust and comprehensive basis for measuring and expressing ESS performance.« less

Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

PubMed

Nguyen, Tien-Thanh; Nguyen, Hoang-Minh; Geiger, Barbara; Mathiesen, Geir; Eijsink, Vincent G H; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

2015-03-07

Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri β-galactosidase. Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant β-galactosidase. The over-expression of recombinant L. reuteri β-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

Nanobarcode gene expression monitoring system for potential miniaturized space applications

NASA Astrophysics Data System (ADS)

Ruan, Weiming; Eastman, P. Scott; Cooke, Patrick A.; Park, Jennifer S.; Chu, Julia S. F.; Gray, Joe W.; Li, Song; Chen, Fanqing Frank

Manned mission to space has been threatened by various cosmos risks including radiation, mirogravity, vacuum, confinement, etc., which may cause genetic variations of astronauts and eventually lead to damages of their health. Thus, the development of small biomedical devices, which can monitor astronaut gene expression changes, is useful for future long-term space missions. Using magnetic microbeads packed with nanocrystal quantum dots at controlled ratios, we were able to generate highly multiplexed nanobarcodes, which can encode a flexible panel of genes. Also, by using a reporter quantum dot, this nanobarcode platform can monitor and quantify gene expression level with improved speed and sensitivity. As a comparison, we studied TGF-β1 induced transcription changes in human bone marrow mesenchymal stem cells with both the nanobarcode microbead system and the Affymetrix GeneChip ® HTA system, which is currently considered as the industrial standard. Though using only 1/20 of the sample RNA, the nanobarcode system showed sensitivity equivalent to Affymetrix GeneChip ® system. The coefficient of variation, dynamic range, and accuracy of the nanobarcodes measurement is equivalent to that of the GeneChip ® HTA system. Therefore, this newly invented nanobarcode microbead platform is thought to be sensitive, flexible, cost-effective and accurate in a level equivalent to the conventional methods. As an extension of the use of this new platform, spacecrafts may carry this miniaturized system as a diagnostic tool for the astronauts.

Three-dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells.

PubMed

Zhan, Kang; Lin, Miao; Liu, MingMei; Sui, YangNan; Babekir, Haitham Mohammed; Zhao, GuoQi

2017-05-01

Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro. © 2016 Japanese Society of Animal Science.

aGEM: an integrative system for analyzing spatial-temporal gene-expression information

PubMed Central

Jiménez-Lozano, Natalia; Segura, Joan; Macías, José Ramón; Vega, Juanjo; Carazo, José María

2009-01-01

Motivation: The work presented here describes the ‘anatomical Gene-Expression Mapping (aGEM)’ Platform, a development conceived to integrate phenotypic information with the spatial and temporal distributions of genes expressed in the mouse. The aGEM Platform has been built by extending the Distributed Annotation System (DAS) protocol, which was originally designed to share genome annotations over the WWW. DAS is a client-server system in which a single client integrates information from multiple distributed servers. Results: The aGEM Platform provides information to answer three main questions. (i) Which genes are expressed in a given mouse anatomical component? (ii) In which mouse anatomical structures are a given gene or set of genes expressed? And (iii) is there any correlation among these findings? Currently, this Platform includes several well-known mouse resources (EMAGE, GXD and GENSAT), hosting gene-expression data mostly obtained from in situ techniques together with a broad set of image-derived annotations. Availability: The Platform is optimized for Firefox 3.0 and it is accessed through a friendly and intuitive display: http://agem.cnb.csic.es Contact: natalia@cnb.csic.es Supplementary information: Supplementary data are available at http://bioweb.cnb.csic.es/VisualOmics/aGEM/home.html and http://bioweb.cnb.csic.es/VisualOmics/index_VO.html and Bioinformatics online. PMID:19592395

Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System.

PubMed

Schwarz-Schilling, Matthaeus; Dupin, Aurore; Chizzolini, Fabio; Krishnan, Swati; Mansy, Sheref S; Simmel, Friedrich C

2018-04-11

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.

PubMed

Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok

2018-03-16

Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.

Optimization of insect cell based protein production processes - online monitoring, expression systems, scale up.

PubMed

Druzinec, Damir; Salzig, Denise; Brix, Alexander; Kraume, Matthias; Vilcinskas, Andreas; Kollewe, Christian; Czermak, Peter

2013-01-01

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes.

Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis.

PubMed

Niedoszytko, M; Bruinenberg, M; van Doormaal, J J; de Monchy, J G R; Nedoszytko, B; Koppelman, G H; Nawijn, M C; Wijmenga, C; Jassem, E; Elberink, J N G Oude

2011-05-01

Anaphylaxis to insect venom (Hymenoptera) is most severe in patients with mastocytosis and may even lead to death. However, not all patients with mastocytosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene expression between patients with indolent systemic mastocytosis (ISM) and a history of insect venom anaphylaxis (IVA) compared to those patients without a history of anaphylaxis, and to determine the predictive use of gene expression profiling. Whole-genome gene expression analysis was performed in peripheral blood cells. Twenty-two adults with ISM were included: 12 with a history of IVA and 10 without a history of anaphylaxis of any kind. Significant differences in single gene expression corrected for multiple testing were found for 104 transcripts (P < 0.05). Gene ontology analysis revealed that the differentially expressed genes were involved in pathways responsible for the development of cancer and focal and cell adhesion suggesting that the expression of genes related to the differentiation state of cells is higher in patients with a history of anaphylaxis. Based on the gene expression profiles, a naïve Bayes prediction model was built identifying patients with IVA. In ISM, gene expression profiles are different between patients with a history of IVA and those without. These findings might reflect a more pronounced mast cells dysfunction in patients without a history of anaphylaxis. Gene expression profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in patients with ISM. Prospective studies are needed to substantiate any conclusions. © 2010 John Wiley & Sons A/S.

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

Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems

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

Conover, David R.; Crawford, Aladsair J.; Viswanathan, Vilayanur V.

2014-06-01

The Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems (PNNL-22010) was first issued in November 2012 as a first step toward providing a foundational basis for developing an initial standard for the uniform measurement and expression of energy storage system (ESS) performance. Its subsequent use in the field and review by the protocol working group and most importantly the users’ subgroup and the thermal subgroup has led to the fundamental modifications reflected in this update of the 2012 Protocol. As an update of the 2012 Protocol, this document (the June 2014 Protocol) is intended to supersedemore » its predecessor and be used as the basis for measuring and expressing ESS performance. The foreword provides general and specific details about what additions, revisions, and enhancements have been made to the 2012 Protocol and the rationale for them in arriving at the June 2014 Protocol.« less

Expression of classical components of the renin-angiotensin system in the human eye.

PubMed

White, Andrew J R; Cheruvu, Sarat C; Sarris, Maria; Liyanage, Surabhi S; Lumbers, Eugenie; Chui, Jeanie; Wakefield, Denis; McCluskey, Peter J

2015-03-01

The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions. © The Author(s) 2014.

[DNA extraction from bones and teeth using AutoMate Express forensic DNA extraction system].

PubMed

Gao, Lin-Lin; Xu, Nian-Lai; Xie, Wei; Ding, Shao-Cheng; Wang, Dong-Jing; Ma, Li-Qin; Li, You-Ying

2013-04-01

To explore a new method in order to extract DNA from bones and teeth automatically. Samples of 33 bones and 15 teeth were acquired by freeze-mill method and manual method, respectively. DNA materials were extracted and quantified from the triturated samples by AutoMate Express forensic DNA extraction system. DNA extraction from bones and teeth were completed in 3 hours using the AutoMate Express forensic DNA extraction system. There was no statistical difference between the two methods in the DNA concentration of bones. Both bones and teeth got the good STR typing by freeze-mill method, and the DNA concentration of teeth was higher than those by manual method. AutoMate Express forensic DNA extraction system is a new method to extract DNA from bones and teeth, which can be applied in forensic practice.

Secretory expression of Lentinula edodes intracellular laccase by yeast high-cell-density system: sub-milligram production of difficult-to-express secretory protein.

PubMed

Kurose, Takeshi; Saito, Yuta; Kimata, Koichi; Nakagawa, Yuko; Yano, Akira; Ito, Keisuke; Kawarasaki, Yasuaki

2014-06-01

While a number of heterologous expression systems have been reported for extracellular laccases, there are few for the intracellular counterparts. The Lentinula edodes intracellular laccase Lcc4 is an industrially potential enzyme with its unique substrate specificity. The heterologous production of the intracellular laccase, however, had been difficult because of its expression-dependent toxicity. We previously demonstrated that recombinant yeast cells synthesized and, interestingly, secreted Lcc4 only when they were suspended to an inducing medium in a high cell-density (J. Biosci. Bioeng., 113, 154-159, 2012). The high cell-density system was versatile and applicable to other difficult-to-express secretory proteins. Nevertheless, the system's great dependence on aeration, which was a practical obstacle to scale-up production of the enzyme and some other proteins, left the secretion pathway and enzymatic properties of the Lcc4 uncharacterized. In this report, we demonstrate a successful production of Lcc4 by applying a jar-fermentor to the high cell-density system. The elevated yield (0.6 mg L(-1)) due to the sufficient aeration allowed us to prepare and purify the enzyme to homogeneity. The enzyme had been secreted as a hyper-glycosylated protein, resulting in smear band-formations in SDS-PAGE. The amino acid sequencing analysis suggested that the N-terminal 17 residues had been recognized as a secretion signal. The recombinant enzyme showed similar enzymatic properties to the naturally occurring Lcc4. The characteristics of the scale-upped expression system, which includes helpful information for the potential users, have also been described. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

VITELLOGENIN EXPRESSION IN SHEEPSHEAD MINNOWS FROM THE PENSACOLA BAY SYSTEM

EPA Science Inventory

Hemmer, M.J., B.L. Hemmer, S.D. Friedman and P.S. Harris. In press. Vitellogenin Expression in Populations of Sheepshead Minnows from the Pensacola Bay System (Abstract). To be presented at the SETAC Fourth World Congress, 14-18 November 2004, Portland, OR. 1 p. (ERL,GB R1015). <...

Upregulation of gene expression in reward-modulatory striatal opioid systems by sleep loss.

PubMed

Baldo, Brian A; Hanlon, Erin C; Obermeyer, William; Bremer, Quentin; Paletz, Elliott; Benca, Ruth M

2013-12-01

Epidemiological studies have shown a link between sleep loss and the obesity 'epidemic,' and several observations indicate that sleep curtailment engenders positive energy balance via increased palatable-food 'snacking.' These effects suggest alterations in reward-modulatory brain systems. We explored the effects of 10 days of sleep deprivation in rats on the expression of striatal opioid peptide (OP) genes that subserve food motivation and hedonic reward, and compared effects with those seen in hypothalamic energy balance-regulatory systems. Sleep-deprived (Sleep-Dep) rats were compared with yoked forced-locomotion apparatus controls (App-Controls), food-restricted rats (Food-Restrict), and unmanipulated controls (Home-Cage). Detection of mRNA levels with in situ hybridization revealed a subregion-specific upregulation of striatal preproenkephalin and prodynorhin gene expression in the Sleep-Dep group relative to all other groups. Neuropeptide Y (NPY) gene expression in the hippocampal dentate gyrus and throughout neocortex was also robustly upregulated selectively in the Sleep-Dep group. In contrast, parallel gene expression changes were observed in the Sleep-Dep and Food-Restrict groups in hypothalamic energy-sensing systems (arcuate nucleus NPY was upregulated, and cocaine- and amphetamine-regulated transcript was downregulated), in alignment with leptin suppression in both groups. Together, these results reveal a novel set of sleep deprivation-induced transcriptional changes in reward-modulatory peptide systems, which are dissociable from the energy-balance perturbations of sleep loss or the potentially stressful effects of the forced-locomotion procedure. The recruitment of telencephalic food-reward systems may provide a feeding drive highly resistant to feedback control, which could engender obesity through the enhancement of palatable feeding.

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

PubMed

Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

2013-01-01

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

Back to basics: pBR322 and protein expression systems in E. coli.

PubMed

Balbás, Paulina; Bolívar, Francisco

2004-01-01

The extensive variety of plasmid-based expression systems in E. coli resulted from the fact that there is no single strategy for achieving maximal expression of every cloned gene. Although a number of strategies have been implemented to deal with problems associated to gene transcription and translation, protein folding, secretion, location, posttranslational modifications, particularities of different strains, and the like and more integrated processes have been developed, the basic plasmid-borne elements and their interaction with the particular host strain will influence the overall expression system and final productivity. Plasmid vector pBR322 is a well-established multipurpose cloning vector in laboratories worldwide, and a large number of derivatives have been created for specific applications and research purposes, including gene expression in its natural host, E. coli, and few other bacteria. The early characterization of the molecule, including its nucleotide sequence, replication and maintenance mechanisms, and determination of its coding regions, accounted for its success, not only as a universal cloning vector, but also as a provider of genes and an origin of replication for other intraspecies vectors. Since the publication of the aforementioned reviews, novel discoveries pertaining to these issues have appeared in the literature that deepen the understanding of the plasmid's features, behavior, and impact in gene expression systems, as well as some important strain characteristics that affect plasmid replication and stability. The objectives of this review include updating and discussing the new information about (1) the replication and maintenance of pBR322; (2) the host-related modulation mechanisms of plasmid replication; (3) the effects of growth rate on replication control, stability, and recombinant gene expression; (4) ways for plasmid amplification and elimination. Finally, (5) a summary of novel ancillary studies about pBR322 is presented.

Expression of hygromycin B resistance in oyster culinary-medicinal mushroom, Pleurotus ostreatus (Jacq.:Fr.)P. Kumm. (higher Basidiomycetes) using three gene expression systems.

PubMed

Dong, Xiaoya; Zhang, Ke; Gao, Yuqian; Qi, Yuancheng; Shen, Jinwen; Qiu, Liyou

2012-01-01

Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster mushroom, Pleurotus ostreatus, plasmid pSHC, pAN7-1, and pBHt1 were evaluated through PEG/CaCl(2)-mediated protoplast transformation. Plasmid pSHC is a newly constructed hph gene expression system, composed of Escherichia coli hph gene, the P. ostreatus sdi promoter, and the CaMV35S terminator. The vector pAN7-1 was commonly used for integrative transformation in filamentous fungi. Plasmid pBHtl is a T-DNA binary vector, usually introduced into fungi by Agrobacterium-mediated transformation. The results showed that plasmids pSHC, pAN7-1, and pBHt1 were all integrated into the host chromosomes and expressed hygromycin B resistance in P. ostreatus. pAN7-1 had the highest transformation efficiency and hph gene expression level, pSHC the second, and pBHt1 the lowest. Growth rates of the transformants on plates containing hygromycin B were in correspondence with their hph gene expression levels. To our knowledge, this is the first report on integrated transformation of plasmid pAN7-1 and pBHt1 in P. ostreatus.

Interaction between sympathetic nervous system and renin angiotensin system on MMPs expression in juvenile rat aorta.

PubMed

Dab, Houcine; Hachani, Rafik; Hodroj, Wassim; Sakly, Mohsen; Bricca, Giampiero; Kacem, Kamel

2011-09-01

The aim of our present study is to investigate the interaction between angiotensin II (ANG II) and sympathetic nervous system (SNS) on matrix metalloproteinase MMP-2 and MMP-9 expression and activity in juvenile rat aorta under normal conditions. Sympathectomy with guanethidine and blockade of the ANG II receptors (AT1R) by losartan were performed alone or in combination on new-born rats. mRNA, protein expression and activity of MMP-2 and MMP-9 were examined by Q-RT-PCR, immunoblotting and zymography, respectively. MMP-2 mRNA and protein amount were decreased after sympathectomy or AT1R blockade and an additive effect was observed after combined treatment. However, MMP-9 expression was reduced to the same level in the three treated groups. There were some detectable gelatinolytic activity of the MMPs in both control and treated rats. We concluded that ANG II stimulates directly and indirectly (via sympathostimulator pathway) the MMP-2 expression but seems unable to affect MMP-9 expression through direct pathway. Combined inhibition of SNS and ANG II were more efficient than a single inhibition in reducing MMP amounts in rat vessels.

Plant expression systems, a budding way to confront chikungunya and Zika in developing countries?

PubMed Central

Cardona-Ospina, Jaime A.; Sepúlveda-Arias, Juan C.; Mancilla, L.; Gutierrez-López, Luis G.

2016-01-01

Plant expression systems could be used as biofactories of heterologous proteins that have the potential to be used with biopharmaceutical aims and vaccine design. This technology is scalable, safe and cost-effective and it has been previously proposed as an option for vaccine and protein pharmaceutical development in developing countries. Here we present a proposal of how plant expression systems could be used to address Zika and chikungunya outbreaks through development of vaccines and rapid diagnostic kits. PMID:27781090

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

PubMed

Birikh, K R; Lebedenko, E N; Boni, I V; Berlin, Y A

1995-10-27

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

Bovine papillomavirus type 4 L1 gene transfection in a Drosophila S2 cell expression system: absence of L1 protein expression

PubMed Central

Góes, Luiz Gustavo Bentim; de Freitas, Antonio Carlos; Ferraz, Oilita Pereira; Rieger, Tania Tassinari; dos Santos, José Ferreira; Pereira, Alexandre; Beçak, Willy; Lindsey, Charles J.; de Cassia Stocco, Rita

2008-01-01

The development of a bovine papillomavirus (BPV) vaccine is an outstanding challenge. BPV protein L1 gene transfection in the Drosophila melanogaster S2 cell expression system failed to produce L1 protein notwithstanding correct L1 gene insertion. Severe genetic inbalance in the host cell line, including cytogenetic alterations, may account for the lack of protein expression. PMID:24031166

A Green-Light-Responsive System for the Control of Transgene Expression in Mammalian and Plant Cells.

PubMed

Chatelle, Claire; Ochoa-Fernandez, Rocio; Engesser, Raphael; Schneider, Nils; Beyer, Hannes M; Jones, Alex R; Timmer, Jens; Zurbriggen, Matias D; Weber, Wilfried

2018-05-18

The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene repression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.

AMTEC radioisotope power system for the Pluto Express mission

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

Ivanenok, J.F. III; Sievers, R.K.

1995-12-31

The Alkali Metal Thermal to Electric Converter (AMTEC) technology has made substantial advances in the last 3 years through design improvements and technical innovations. In 1993 programs began to produce an AMTEC cell specifically for the NASA Pluto Express Mission. A set of efficiency goals was established for this series of cells to be developed. According to this plan, cell {number_sign}8 would be 17% efficient but was actually 18% efficient. Achieving this goal, as well as design advances that allow the cell to be compact, has resulted in pushing the cell from an unexciting 2 W/kg and 2% efficiency tomore » very attractive 40 W/kg and 18% measured efficiency. This paper will describe the design and predict the performance of a radioisotope powered AMTEC system for the Pluto Express mission.« less

Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems.

PubMed

Westhoff, C M; Wylie, D E

2006-01-01

The development and use of heterologous expression systems is critical for deciphering the function of mammalian Rh and Rh-glycoproteins. The studies here use Xenopus oocytes, well known for their ability to readily traffic and express difficult membrane proteins, and S. cerevisiae wild-type strains and mutants that are defective in ammonium transport. Data obtained in both of these expression systems revealed that mammalian Rh-glycoprotein-mediated transport (RhAG, RhBG, and RhCG) is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient, effectively exchanging NH4+ for H+ in a process that results in transport of net NH3. Homology modeling and functional studies suggest that the more recently evolved erythrocyte blood group proteins, RhCE and RhD, may not function directly in ammonia transport and may be evolving a new function in the RBC membrane. The relationship of Rh and Rh-glycoproteins to the Amt/Mep ammonium transporters is substantiated with functional transport data and structural modeling.

Expression of VGF mRNA in the adult rat central nervous system.

PubMed

Snyder, S E; Salton, S R

1998-04-27

VGF is a secretory peptide precursor that is expressed and processed by neuronal cells in a cell type-specific fashion. In addition, VGF transcription and secretion are rapidly and relatively selectively induced by neurotrophins and depolarization in vitro. To gain insight into the possible function(s) of VGF in the nervous system, we have carried out a detailed examination of the distribution of VGF mRNA in the adult rat central nervous system by using in situ hybridization. Robust expression was detected in many neurons throughout the brain and spinal cord, in several types of neurons in the retina, and in presumptive chromaffin cells of the adrenal medulla. In the brain, prominent expression of VGF mRNA was observed in neurons of the main and accessory olfactory bulbs; in the anterior olfactory nucleus; in the induseum griseum and taenia tecta; in the olfactory tubercle; in CA1-CA3, the hilus of the dentate gyrus, and the subicular complex of the hippocampal formation; in the piriform, periamygdaloid, transitional, and lateral entorhinal cortices; in the endopiriform nucleus; in the hypothalamus, particularly the preoptic, periventricular, supraoptic, suprachiasmatic, and arcuate nuclei; and in a number of septal, thalamic, amygdaloid, and brainstem nuclei. Labeling was also seen in neurons of the neocortex and transitional cortical areas, particularly in layer V, and in basal ganglia and cerebellum. These data demonstrate that VGF mRNA is expressed much more extensively in the brain than has been described in previous RNA or immunohistochemical studies, and, furthermore, that VGF is widely expressed in the spinal cord and retina.

Expression of RYamide in the nervous and endocrine system of Bombyx mori.

PubMed

Roller, Ladislav; Čižmár, Daniel; Bednár, Branislav; Žitňan, Dušan

2016-06-01

RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12-14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression and Function of the Cholinergic System in Immune Cells

PubMed Central

Fujii, Takeshi; Mashimo, Masato; Moriwaki, Yasuhiro; Misawa, Hidemi; Ono, Shiro; Horiguchi, Kazuhide; Kawashima, Koichiro

2017-01-01

T and B cells express most cholinergic system components—e.g., acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase, and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Using ChATBAC-eGFP transgenic mice, ChAT expression has been confirmed in T and B cells, dendritic cells, and macrophages. Moreover, T cell activation via T-cell receptor/CD3-mediated pathways upregulates ChAT mRNA expression and ACh synthesis, suggesting that this lymphocytic cholinergic system contributes to the regulation of immune function. Immune cells express all five mAChRs (M1–M5). Combined M1/M5 mAChR-deficient (M1/M5-KO) mice produce less antigen-specific antibody than wild-type (WT) mice. Furthermore, spleen cells in M1/M5-KO mice produce less tumor necrosis factor (TNF)-α and interleukin (IL)-6, suggesting M1/M5 mAChRs are involved in regulating pro-inflammatory cytokine and antibody production. Immune cells also frequently express the α2, α5, α6, α7, α9, and α10 nAChR subunits. α7 nAChR-deficient (α7-KO) mice produce more antigen-specific antibody than WT mice, and spleen cells from α7-KO mice produce more TNF-α and IL-6 than WT cells. This suggests that α7 nAChRs are involved in regulating cytokine production and thus modulate antibody production. Evidence also indicates that nicotine modulates immune responses by altering cytokine production and that α7 nAChR signaling contributes to immunomodulation through modification of T cell differentiation. Together, these findings suggest the involvement of both mAChRs and nAChRs in the regulation of immune function. The observation that vagus nerve stimulation protects mice from lethal endotoxin shock led to the notion of a cholinergic anti-inflammatory reflex pathway, and the spleen is an essential component of this anti-inflammatory reflex. Because the spleen lacks direct vagus innervation, it has been postulated that ACh synthesized by a subset of CD4+ T cells relays

Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts.

PubMed

Wang, Weishan; Yang, Tongjian; Li, Yihong; Li, Shanshan; Yin, Shouliang; Styles, Kathryn; Corre, Christophe; Yang, Keqian

2016-07-15

Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.7 × 10(-10) M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01-4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes.

Stability of Lentiviral Vector-Mediated Transgene Expression in the Brain in the Presence of Systemic Antivector Immune Responses

PubMed Central

ABORDO-ADESIDA, EVELYN; FOLLENZI, ANTONIA; BARCIA, CARLOS; SCIASCIA, SANDRA; CASTRO, MARIA G.; NALDINI, LUIGI; LOWENSTEIN, PEDRO R.

2009-01-01

Lentiviral vectors are promising tools for gene therapy in the CNS. It is therefore important to characterize their interactions with the immune system in the CNS. This work characterizes transgene expression and brain inflammation in the presence or absence of immune responses generated after systemic immunization with lentiviral vectors. We characterized transduction with SIN-LV vectors in the CNS. A dose—response curve using SIN-LV-GFP demonstrated detectable transgene expression in the striatum at a dose of 102, and maximum expression at 106, transducing units of lentiviral vector, with minimal increase in inflammatory markers between the lowest and highest dose of vector injected. Our studies demonstrate that injection of a lentiviral vector into the CNS did not cause a measurable inflammatory response. Systemic immunization after CNS injection, with the lentiviral vector expressing the same transgene as a vector injected into the CNS, caused a decrease in transgene expression in the CNS, concomitantly with an infiltration of inflammatory cells into the CNS parenchyma at the injection site. However, peripheral immunization with a lentiviral vector carrying a different transgene did not diminish transgene expression, or cause CNS inflammation. Systemic immunization preceding injection of lentiviral vectors into the CNS determined that preexisting antilentiviral immunity, regardless of the transgene, did not affect transgene expression. Furthermore, we showed that the transgene, but not the virion or vector components, is responsible for providing antigenic epitopes to the activated immune system, on systemic immunization with lentivirus. Low immunogenicity and prolonged transgene expression in the presence of preexisting lentiviral immunity are encouraging data for the future use of lentiviral vectors in CNS gene therapy. In summary, the lentiviral vectors tested induced undetectable activation of innate immune responses, and stimulation of adaptive immune

[Facial expressions of negative emotions in clinical interviews: The development, reliability and validity of a categorical system for the attribution of functions to facial expressions of negative emotions].

PubMed

Bock, Astrid; Huber, Eva; Peham, Doris; Benecke, Cord

2015-01-01

The development (Study 1) and validation (Study 2) of a categorical system for the attribution of facial expressions of negative emotions to specific functions. The facial expressions observed inOPDinterviews (OPD-Task-Force 2009) are coded according to the Facial Action Coding System (FACS; Ekman et al. 2002) and attributed to categories of basic emotional displays using EmFACS (Friesen & Ekman 1984). In Study 1 we analyze a partial sample of 20 interviews and postulate 10 categories of functions that can be arranged into three main categories (interactive, self and object). In Study 2 we rate the facial expressions (n=2320) from the OPD interviews (10 minutes each interview) of 80 female subjects (16 healthy, 64 with DSM-IV diagnosis; age: 18-57 years) according to the categorical system and correlate them with problematic relationship experiences (measured with IIP,Horowitz et al. 2000). Functions of negative facial expressions can be attributed reliably and validly with the RFE-Coding System. The attribution of interactive, self-related and object-related functions allows for a deeper understanding of the emotional facial expressions of patients with mental disorders.

A simple and robust vector-based shRNA expression system used for RNA interference.

PubMed

Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

2013-01-01

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

mRNA expression of CDH3, IGF2BP3, and BIRC5 in biliary brush cytology specimens is a useful adjunctive tool of cytology for the diagnosis of malignant biliary stricture

PubMed Central

Kim, Tae Ho; Chang, Jae Hyuck; Lee, Hee Jin; Kim, Jean A; Lim, Yeon Soo; Kim, Chang Whan; Han, Sok Won

2016-01-01

Abstract Although advances have been made in diagnostic tools, the distinction between malignant and benign biliary strictures still remains challenging. Intraductal brush cytology is a convenient and safe method that is used for the diagnosis of biliary stricture, but, low sensitivity limits its usefulness. This study aimed to demonstrate the usefulness of mRNA expression levels of target genes in brush cytology specimens combined with cytology for the diagnosis of malignant biliary stricture. Immunohistochemistry for cadherin 3 (CDH3), p53, insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3), homeobox B7 (HOXB7), and baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) was performed in 4 benign and 4 malignant bile duct tissues. Through endoscopic or interventional radiologic procedures, brush cytology specimens were prospectively obtained in 21 and 35 paitents with biliary strictures. In the brush cytology specimens, the mRNA expressions levels of 5 genes were determined by real-time polymerase chain reaction. Immunohistochemistry for CDH3, p53, IGF2BP3, HOXB7, and BIRC5 all showed positive staining in malignant tissues in contrast to benign tissues, which were negative. In the brush cytology specimens, the mRNA expression levels of CDH3, IGF2BP3, HOXB7, and BIRC5 were significantly higher in cases of malignant biliary stricture compared with cases of benign stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.001). The receiver-operating characteristic curves of these 4 mRNAs demonstrated that mRNA expression levels are useful for the prediction of malignant biliary stricture (P = 0.006, P < 0.001, P < 0.001, and P = 0.002). The sensitivity and specificity, respectively, for malignant biliary stricture were 57.1% and 100% for cytology, 57.1% and 64.3% for CDH3, 76.2% and 100% for IGF2BP3, 71.4% and 57.1% for HOXB7, and 76.2% and 64.3% for BIRC5. When cytology was combined with the mRNA levels of CDH3, IGF2BP3, or BIRC5, the

Integrating T7 RNA Polymerase and Its Cognate Transcriptional Units for a Host-Independent and Stable Expression System in Single Plasmid.

PubMed

Liang, Xiao; Li, Chenmeng; Wang, Wenya; Li, Qiang

2018-05-18

Metabolic engineering and synthetic biology usually require universal expression systems for stable and efficient gene expression in various organisms. In this study, a host-independent and stable T7 expression system had been developed by integrating T7 RNA polymerase and its cognate transcriptional units in single plasmid. The expression of T7 RNA polymerase was restricted below its lethal threshold using a T7 RNA polymerase antisense gene cassette, which allowed long periods of cultivation and protein production. In addition, by designing ribosome binding sites, we further tuned the expression capacity of this novel T7 system within a wide range. This host-independent expression system efficiently expressed genes in five different Gram-negative strains and one Gram-positive strain and was also shown to be applicable in a real industrial d- p-hydroxyphenylglycine production system.

Systems for the expression of orthogonal translation components eubacterial host cells

DOEpatents

Ryu, Youngha [San Diego, CA; Schultz, Peter G [La Jolla, CA

2012-06-12

The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.

PubMed

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-03-06

Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely

A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection.

PubMed

Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L; Chan, Leanne Jade G; Wei, Tong; Joe, Anna; Thomas, Nicholas; Pruitt, Rory; Adams, Paul D; Chern, Maw Sheng; Petzold, Christopher J; Liu, Chang C; Ronald, Pamela C

2016-04-18

Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.

A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection

DOE PAGES

Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L.; ...

2015-11-27

Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.

Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system.

PubMed

Kofuku, Yutaka; Yokomizo, Tomoki; Imai, Shunsuke; Shiraishi, Yutaro; Natsume, Mei; Itoh, Hiroaki; Inoue, Masayuki; Nakata, Kunio; Igarashi, Shunsuke; Yamaguchi, Hideyuki; Mizukoshi, Toshimi; Suzuki, Ei-Ichiro; Ueda, Takumi; Shimada, Ichio

2018-03-08

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl- 13 C 1 H 3 -labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β 2 -adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

A real-time control system of gene expression using ligand-bound nucleic acid aptamer for metabolic engineering.

PubMed

Wang, Jing; Cui, Xun; Yang, Le; Zhang, Zhe; Lv, Liping; Wang, Haoyuan; Zhao, Zhenmin; Guan, Ningzi; Dong, Lichun; Chen, Rachel

2017-07-01

Artificial control of bio-functions through regulating gene expression is one of the most important and attractive technologies to build novel living systems that are useful in the areas of chemical synthesis, nanotechnology, pharmacology, cell biology. Here, we present a novel real-time control system of gene regulation that includes an enhancement element by introducing duplex DNA aptamers upstream promoter and a repression element by introducing a RNA aptamer upstream ribosome binding site. With the presence of ligands corresponding to the DNA aptamers, the expression of the target gene can be potentially enhanced at the transcriptional level by strengthening the recognition capability of RNAP to the recognition region and speeding up the separation efficiency of the unwinding region due to the induced DNA bubble around the thrombin-bound aptamers; while with the presence of RNA aptamer ligand, the gene expression can be repressed at the translational level by weakening the recognition capability of ribosome to RBS due to the shielding of RBS by the formed aptamer-ligand complex upstream RBS. The effectiveness and potential utility of the developed gene regulation system were demonstrated by regulating the expression of ecaA gene in the cell-free systems. The realistic metabolic engineering application of the system has also tested by regulating the expression of mgtC gene and thrombin cDNA in Escherichia coli JD1021 for controlling metabolic flux and improving thrombin production, verifying that the real-time control system of gene regulation is able to realize the dynamic regulation of gene expression with potential applications in bacterial physiology studies and metabolic engineering. Copyright © 2017. Published by Elsevier Inc.

EXPRESS Rack Mockup

NASA Technical Reports Server (NTRS)

2002-01-01

The EXPRESS Rack is a standardized payload rack system that transports, stores, and supports experiments aboard the International Space Station (ISS). EXPRESS stands for EXpedite the PRocessing of Experiments to the Space Station, reflecting the fact that this system was developed specifically to maximize the Station's research capabilities. The EXPRESS Rack system supports science payloads in several disciplines, including biology, chemistry, physics, ecology, and medicine. With the EXPRESS Rack, getting experiments to space has never been easier or more affordable. With its standardized hardware interfaces and streamlined approach, the EXPRESS Rack enables quick, simple integration of multiple payloads aboard the ISS. The system is comprised of elements that remain on the ISS, as well as elements that travel back and forth between the ISS and Earth via the Space Shuttle. The Racks stay on orbit continually, while experiments are exchanged in and out of the EXPRESS Racks as needed, remaining on the ISS for three months to several years, depending on the experiment's time requirements. A refrigerator-sized Rack can be divided into segments, as large as half of an entire rack or as small as a bread box. Payloads within EXPRESS Racks can operate independently of each other, allowing for differences in temperature, power levels, and schedules. Experiments contained within EXPRESS Racks may be controlled by the ISS crew or remotely by the Payload Rack Officer at the Payload Operations Center at the Marshall Space Flight Center (MSFC). The EXPRESS Rack system was developed by MSFC and built by the Boeing Co. in Huntsville, Alabama. Eight EXPRESS Racks are being built for use on the ISS.

Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange.

PubMed

Matsunari, Hitomi; Onodera, Masafumi; Tada, Norihiro; Mochizuki, Hideki; Karasawa, Satoshi; Haruyama, Erika; Nakayama, Naoki; Saito, Hitoshi; Ueno, Satoshi; Kurome, Mayuko; Miyawaki, Atsushi; Nagashima, Hiroshi

2008-09-01

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector D Delta Nsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.

Understanding the Earth Systems: Expressions of Dynamic and Cyclic Thinking among University Students

ERIC Educational Resources Information Center

Batzri, Or; Ben Zvi Assaraf, Orit; Cohen, Carmit; Orion, Nir

2015-01-01

In this two-part study, we examine undergraduate university students' expression of two important system thinking characteristics--dynamic thinking and cyclic thinking--focusing particularly on students of geology. The study was conducted using an Earth systems questionnaire designed to elicit and reflect either dynamic or cyclic thinking. The…

BioVLAB-mCpG-SNP-EXPRESS: A system for multi-level and multi-perspective analysis and exploration of DNA methylation, sequence variation (SNPs), and gene expression from multi-omics data.

PubMed

Chae, Heejoon; Lee, Sangseon; Seo, Seokjun; Jung, Daekyoung; Chang, Hyeonsook; Nephew, Kenneth P; Kim, Sun

2016-12-01

Measuring gene expression, DNA sequence variation, and DNA methylation status is routinely done using high throughput sequencing technologies. To analyze such multi-omics data and explore relationships, reliable bioinformatics systems are much needed. Existing systems are either for exploring curated data or for processing omics data in the form of a library such as R. Thus scientists have much difficulty in investigating relationships among gene expression, DNA sequence variation, and DNA methylation using multi-omics data. In this study, we report a system called BioVLAB-mCpG-SNP-EXPRESS for the integrated analysis of DNA methylation, sequence variation (SNPs), and gene expression for distinguishing cellular phenotypes at the pairwise and multiple phenotype levels. The system can be deployed on either the Amazon cloud or a publicly available high-performance computing node, and the data analysis and exploration of the analysis result can be conveniently done using a web-based interface. In order to alleviate analysis complexity, all the process are fully automated, and graphical workflow system is integrated to represent real-time analysis progression. The BioVLAB-mCpG-SNP-EXPRESS system works in three stages. First, it processes and analyzes multi-omics data as input in the form of the raw data, i.e., FastQ files. Second, various integrated analyses such as methylation vs. gene expression and mutation vs. methylation are performed. Finally, the analysis result can be explored in a number of ways through a web interface for the multi-level, multi-perspective exploration. Multi-level interpretation can be done by either gene, gene set, pathway or network level and multi-perspective exploration can be explored from either gene expression, DNA methylation, sequence variation, or their relationship perspective. The utility of the system is demonstrated by performing analysis of phenotypically distinct 30 breast cancer cell line data set. BioVLAB-mCpG-SNP-EXPRESS is

An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

PubMed

Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

2015-09-01

A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

Self-Expression or Teacher Influence: The Shaw System of Finger-Painting.

ERIC Educational Resources Information Center

Stankiewicz, Mary Ann

1984-01-01

Finger painting is often regarded as the epitome of free expression for children. However, a careful review of the history of Ruth Shaw's finger-painting system reveals that it was dominated by specific techniques and stylistic conventions taught without a critical understanding of art history or appreciation. (IS)

The Effect of an Intelligent Tutoring System (ITS) on Student Achievement in Algebraic Expression

ERIC Educational Resources Information Center

Chien, Tsai Chen; Md. Yunus, Aida Suraya; Ali, Wan Zah Wan; Bakar, Ab. Rahim

2008-01-01

In this experimental study, use of Computer Assisted Instruction (CAI) followed by use of an Intelligent Tutoring System (CAI+ITS) was compared to the use of CAI (CAI only) in tutoring students on the topic of Algebraic Expression. Two groups of students participated in the study. One group of 32 students studied algebraic expression in a CAI…

Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression

PubMed Central

Puthiyaveetil, Sujith; Allen, John F.

2009-01-01

Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles—chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems. PMID:19324807

Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression.

PubMed

Puthiyaveetil, Sujith; Allen, John F

2009-06-22

Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles-chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems.

Selenium regulates gene expression of selenoprotein W in chicken skeletal muscle system.

PubMed

Ruan, Hongfeng; Zhang, Ziwei; Wu, Qiong; Yao, Haidong; Li, Jinlong; Li, Shu; Xu, Shiwen

2012-01-01

Selenoprotein W (SelW) is abundantly expressed in skeletal muscles of mammals and necessary for the metabolism of skeletal muscles. However, its expression pattern in skeletal muscle system of birds is still uncovered. Herein, to investigate the distribution of SelW mRNA in chicken skeletal muscle system and its response to different selenium (Se) status, 1-day-old chickens were exposed to various concentrations of Se as sodium selenite in the feed for 35 days. In addition, myoblasts were treated with different concentrations of Se in the medium for 72 h. Then the levels of SelW mRNA in skeletal muscles (wing muscle, pectoral muscle, thigh muscle) and myoblasts were determined on days 1, 15, 25, and 35 and at 0, 24, 48, and 72 h, respectively. The results showed that SelW was detected in all these muscle components and it increased both along with the growth of organism and the differentiation process of myoblasts. The thigh muscle is more responsive to Se intake than the other two skeletal muscle tissues while the optimal Se supplementation for SelW mRNA expression in chicken myoblasts was 10(-7) M. In summary, Se plays important roles in the development of chicken skeletal muscles. To effect optimal SelW gene expression, Se must be provided in the diet and the media in adequate amounts and neither at excessive nor deficient levels.

Gene expression-based molecular diagnostic system for malignant gliomas is superior to histological diagnosis.

PubMed

Shirahata, Mitsuaki; Iwao-Koizumi, Kyoko; Saito, Sakae; Ueno, Noriko; Oda, Masashi; Hashimoto, Nobuo; Takahashi, Jun A; Kato, Kikuya

2007-12-15

Current morphology-based glioma classification methods do not adequately reflect the complex biology of gliomas, thus limiting their prognostic ability. In this study, we focused on anaplastic oligodendroglioma and glioblastoma, which typically follow distinct clinical courses. Our goal was to construct a clinically useful molecular diagnostic system based on gene expression profiling. The expression of 3,456 genes in 32 patients, 12 and 20 of whom had prognostically distinct anaplastic oligodendroglioma and glioblastoma, respectively, was measured by PCR array. Next to unsupervised methods, we did supervised analysis using a weighted voting algorithm to construct a diagnostic system discriminating anaplastic oligodendroglioma from glioblastoma. The diagnostic accuracy of this system was evaluated by leave-one-out cross-validation. The clinical utility was tested on a microarray-based data set of 50 malignant gliomas from a previous study. Unsupervised analysis showed divergent global gene expression patterns between the two tumor classes. A supervised binary classification model showed 100% (95% confidence interval, 89.4-100%) diagnostic accuracy by leave-one-out cross-validation using 168 diagnostic genes. Applied to a gene expression data set from a previous study, our model correlated better with outcome than histologic diagnosis, and also displayed 96.6% (28 of 29) consistency with the molecular classification scheme used for these histologically controversial gliomas in the original article. Furthermore, we observed that histologically diagnosed glioblastoma samples that shared anaplastic oligodendroglioma molecular characteristics tended to be associated with longer survival. Our molecular diagnostic system showed reproducible clinical utility and prognostic ability superior to traditional histopathologic diagnosis for malignant glioma.

Systems Reconsolidation Reveals a Selective Role for the Anterior Cingulate Cortex in Generalized Contextual Fear Memory Expression

PubMed Central

Einarsson, Einar Ö; Pors, Jennifer; Nader, Karim

2015-01-01

After acquisition, hippocampus-dependent memories undergo a systems consolidation process, during which they become independent of the hippocampus and dependent on the anterior cingulate cortex (ACC) for memory expression. However, consolidated remote memories can become transiently hippocampus-dependent again following memory reactivation. How this systems reconsolidation affects the role of the ACC in remote memory expression is not known. Using contextual fear conditioning, we show that the expression of 30-day-old remote memory can transiently be supported by either the ACC or the dorsal hippocampus following memory reactivation, and that the ACC specifically mediates expression of remote generalized contextual fear memory. We found that suppression of neural activity in the ACC with the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) impaired the expression of remote, but not recent, contextual fear memory. Fear expression was not affected by this treatment if preceded by memory reactivation 6 h earlier, nor was it affected by suppression of neural activity in the dorsal hippocampus with the GABA-receptor agonist muscimol. However, simultaneous targeting of both the ACC and the dorsal hippocampus 6 h after memory reactivation disrupted contextual fear memory expression. Second, we observed that expression of a 30-day-old generalized contextual fear memory in a novel context was not affected by memory reactivation 6 h earlier. However, intra-ACC CNQX infusion before testing impaired contextual fear expression in the novel context, but not the original training context. Together, these data suggest that although the dorsal hippocampus may be recruited during systems reconsolidation, the ACC remains necessary for the expression of generalized contextual fear memory. PMID:25091528

Systems reconsolidation reveals a selective role for the anterior cingulate cortex in generalized contextual fear memory expression.

PubMed

Einarsson, Einar Ö; Pors, Jennifer; Nader, Karim

2015-01-01

After acquisition, hippocampus-dependent memories undergo a systems consolidation process, during which they become independent of the hippocampus and dependent on the anterior cingulate cortex (ACC) for memory expression. However, consolidated remote memories can become transiently hippocampus-dependent again following memory reactivation. How this systems reconsolidation affects the role of the ACC in remote memory expression is not known. Using contextual fear conditioning, we show that the expression of 30-day-old remote memory can transiently be supported by either the ACC or the dorsal hippocampus following memory reactivation, and that the ACC specifically mediates expression of remote generalized contextual fear memory. We found that suppression of neural activity in the ACC with the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) impaired the expression of remote, but not recent, contextual fear memory. Fear expression was not affected by this treatment if preceded by memory reactivation 6 h earlier, nor was it affected by suppression of neural activity in the dorsal hippocampus with the GABA-receptor agonist muscimol. However, simultaneous targeting of both the ACC and the dorsal hippocampus 6 h after memory reactivation disrupted contextual fear memory expression. Second, we observed that expression of a 30-day-old generalized contextual fear memory in a novel context was not affected by memory reactivation 6 h earlier. However, intra-ACC CNQX infusion before testing impaired contextual fear expression in the novel context, but not the original training context. Together, these data suggest that although the dorsal hippocampus may be recruited during systems reconsolidation, the ACC remains necessary for the expression of generalized contextual fear memory.

High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.

PubMed

Zhang, Kang; Su, Lingqia; Duan, Xuguo; Liu, Lina; Wu, Jing

2017-02-20

We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, P amyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536. Using the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the P amyQ' promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter P HpaII -P amyQ' produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter P HpaII -P amyQ' also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter P HpaII -P amyQ' system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications. The dual-promoter P HpaII -P amyQ' system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

System for face recognition under expression variations of neutral-sampled individuals using recognized expression warping and a virtual expression-face database

NASA Astrophysics Data System (ADS)

Petpairote, Chayanut; Madarasmi, Suthep; Chamnongthai, Kosin

2018-01-01

The practical identification of individuals using facial recognition techniques requires the matching of faces with specific expressions to faces from a neutral face database. A method for facial recognition under varied expressions against neutral face samples of individuals via recognition of expression warping and the use of a virtual expression-face database is proposed. In this method, facial expressions are recognized and the input expression faces are classified into facial expression groups. To aid facial recognition, the virtual expression-face database is sorted into average facial-expression shapes and by coarse- and fine-featured facial textures. Wrinkle information is also employed in classification by using a process of masking to adjust input faces to match the expression-face database. We evaluate the performance of the proposed method using the CMU multi-PIE, Cohn-Kanade, and AR expression-face databases, and we find that it provides significantly improved results in terms of face recognition accuracy compared to conventional methods and is acceptable for facial recognition under expression variation.

Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus.

PubMed

Wiley, Kenneth L; Treadwell, Edward; Manigaba, Kayihura; Word, Beverly; Lyn-Cook, Beverly D

2013-02-01

Systemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy. Real-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlash(TM) methylated quantification colorimetric assay. Significant increase in DNMT3A (p < 0.001) was shown in lupus patients when compared to age-matched healthy controls. This increase was associated with a higher SLEDI index. More striking was that expression levels for African American (AA) women were higher than European American women in the lupus populations. A subset of AA women on DHEA therapy showed a significant decrease (p < 0.05) in DNMT3A expression in comparison to lupus patients not on the therapy. DHEA is an androgenic steroid found in low levels in the serum of lupus patients. Supplementation of this hormone has been shown to be beneficial to some lupus patients. DHEA was not shown to effect DNMT1 or DNMT3B expression. Increased expression was also noted in DNMT3B (p < 0.05) in lupus patients compared to age-matched healthy controls. However, no significant difference was noted in DNMT1 (p = 0.2148) expression between lupus patients and healthy controls. Although increases were detected in de novo methyltransferases, a global decrease (p < 0.001) in 5-methycytosine was observed in

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.

Increasing the yield of middle silk gland expression system through transgenic knock-down of endogenous sericin-1.

PubMed

Ma, Sanyuan; Xia, Xiaojuan; Li, Yufeng; Sun, Le; Liu, Yue; Liu, Yuanyuan; Wang, Xiaogang; Shi, Run; Chang, Jiasong; Zhao, Ping; Xia, Qingyou

2017-08-01

Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.

Role of endogenous cortistatin in the regulation of ghrelin system expression at pancreatic level under normal and obese conditions.

PubMed

Chanclón, Belén; Luque, Raúl M; Córdoba-Chacón, José; Gahete, Manuel D; Pozo-Salas, Ana I; Castaño, Justo P; Gracia-Navarro, Francisco; Martínez-Fuentes, Antonio J

2013-01-01

Ghrelin-system components [native ghrelin, In1-ghrelin, Ghrelin-O-acyltransferase enzyme (GOAT) and receptors (GHS-Rs)] are expressed in a wide variety of tissues, including the pancreas, where they exert different biological actions including regulation of neuroendocrine secretions, food intake and pancreatic function. The expression of ghrelin system is regulated by metabolic conditions (fasting/obesity) and is associated with the progression of obesity and insulin resistance. Cortistatin (CORT), a neuropeptide able to activate GHS-R, has emerged as an additional link in gut-brain interplay. Indeed, we recently reported that male CORT deficient mice (cort-/-) are insulin-resistant and present a clear dysregulation in the stomach ghrelin-system. The present work was focused at analyzing the expression pattern of ghrelin-system components at pancreas level in cort-/- mice and their control littermates (cort +/+) under low- or high-fat diet. Our data reveal that all the ghrelin-system components are expressed at the mouse pancreatic level, where, interestingly, In1-ghrelin was expressed at higher levels than native-ghrelin. Thus, GOAT mRNA levels were significantly lower in cort-/- mice compared with controls while native ghrelin, In1-ghrelin and GHS-R transcript levels remained unaltered under normal metabolic conditions. Moreover, under obese condition, a significant increase in pancreatic expression of native-ghrelin, In1-ghrelin and GHS-R was observed in obese cort+/+ but not in cort-/- mice. Interestingly, insulin expression and release was elevated in obese cort+/+, while these changes were not observed in obese cort-/- mice. Altogether, our results indicate that the ghrelin-system expression is clearly regulated in the pancreas of cort+/+ and cort -/- under normal and/or obesity conditions suggesting that this system may play relevant roles in the endocrine pancreas. Most importantly, our data demonstrate, for the first time, that endogenous CORT is essential

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

Adipose tissue endocannabinoid system gene expression: depot differences and effects of diet and exercise

PubMed Central

2011-01-01

Background Alterations of endocannabinoid system in adipose tissue play an important role in lipid regulation and metabolic dysfunction associated with obesity. The purpose of this study was to determine whether gene expression levels of cannabinoid type 1 receptor (CB1) and fatty acid amide hydrolase (FAAH) are different in subcutaneous abdominal and gluteal adipose tissue, and whether hypocaloric diet and aerobic exercise influence subcutaneous adipose tissue CB1 and FAAH gene expression in obese women. Methods Thirty overweight or obese, middle-aged women (BMI = 34.3 ± 0.8 kg/m2, age = 59 ± 1 years) underwent one of three 20-week weight loss interventions: caloric restriction only (CR, N = 9), caloric restriction plus moderate-intensity aerobic exercise (CRM, 45-50% HRR, N = 13), or caloric restriction plus vigorous-intensity aerobic exercise (CRV, 70-75% HRR, N = 8). Subcutaneous abdominal and gluteal adipose tissue samples were collected before and after the interventions to measure CB1 and FAAH gene expression. Results At baseline, FAAH gene expression was higher in abdominal, compared to gluteal adipose tissue (2.08 ± 0.11 vs. 1.78 ± 0.10, expressed as target gene/β-actin mRNA ratio × 10-3, P < 0.05). Compared to pre-intervention, CR did not change abdominal, but decreased gluteal CB1 (Δ = -0.82 ± 0.25, P < 0.05) and FAAH (Δ = -0.49 ± 0.14, P < 0.05) gene expression. CRM or CRV alone did not change adipose tissue CB1 and FAAH gene expression. However, combined CRM and CRV (CRM+CRV) decreased abdominal adipose tissue FAAH gene expression (Δ = -0.37 ± 0.18, P < 0.05). The changes in gluteal CB1 and abdominal FAAH gene expression levels in the CR alone and the CRM+CRV group were different (P < 0.05) or tended to be different (P = 0.10). Conclusions There are depot differences in subcutaneous adipose tissue endocannabinoid system gene expression in obese individuals. Aerobic exercise training may preferentially modulate abdominal adipose tissue

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles.

PubMed

Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J; Chen, Qiang; Mason, Hugh S

2008-03-28

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

PubMed Central

Santi, Luca; Batchelor, Lance; Huang, Zhong; Hjelm, Brooke; Kilbourne, Jacquelyn; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

2009-01-01

Virus like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNV) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post infection. Oral immunization of CD1 mice with 100 or 250 μg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic. PMID:18325641

Maintenance and expression of the S. cerevisiae mitochondrial genome--from genetics to evolution and systems biology.

PubMed

Lipinski, Kamil A; Kaniak-Golik, Aneta; Golik, Pawel

2010-01-01

As a legacy of their endosymbiotic eubacterial origin, mitochondria possess a residual genome, encoding only a few proteins and dependent on a variety of factors encoded by the nuclear genome for its maintenance and expression. As a facultative anaerobe with well understood genetics and molecular biology, Saccharomyces cerevisiae is the model system of choice for studying nucleo-mitochondrial genetic interactions. Maintenance of the mitochondrial genome is controlled by a set of nuclear-coded factors forming intricately interconnected circuits responsible for replication, recombination, repair and transmission to buds. Expression of the yeast mitochondrial genome is regulated mostly at the post-transcriptional level, and involves many general and gene-specific factors regulating splicing, RNA processing and stability and translation. A very interesting aspect of the yeast mitochondrial system is the relationship between genome maintenance and gene expression. Deletions of genes involved in many different aspects of mitochondrial gene expression, notably translation, result in an irreversible loss of functional mtDNA. The mitochondrial genetic system viewed from the systems biology perspective is therefore very fragile and lacks robustness compared to the remaining systems of the cell. This lack of robustness could be a legacy of the reductive evolution of the mitochondrial genome, but explanations involving selective advantages of increased evolvability have also been postulated. Copyright © 2009 Elsevier B.V. All rights reserved.

Pregnancy-Induced Changes in Systemic Gene Expression among Healthy Women and Women with Rheumatoid Arthritis

PubMed Central

Mittal, Anuradha; Pachter, Lior; Nelson, J. Lee; Smed, Mette Kiel; Gildengorin, Virginia L.; Zoffmann, Vibeke; Hetland, Merete Lund; Jewell, Nicholas P.; Olsen, Jørn; Jawaheer, Damini

2015-01-01

Background Pregnancy induces drastic biological changes systemically, and has a beneficial effect on some autoimmune conditions such as rheumatoid arthritis (RA). However, specific systemic changes that occur as a result of pregnancy have not been thoroughly examined in healthy women or women with RA. The goal of this study was to identify genes with expression patterns associated with pregnancy, compared to pre-pregnancy as baseline and determine whether those associations are modified by presence of RA. Results In our RNA sequencing (RNA-seq) dataset from 5 healthy women and 20 women with RA, normalized expression levels of 4,710 genes were significantly associated with pregnancy status (pre-pregnancy, first, second and third trimesters) over time, irrespective of presence of RA (False Discovery Rate (FDR)-adjusted p value<0.05). These genes were enriched in pathways spanning multiple systems, as would be expected during pregnancy. A subset of these genes (n = 256) showed greater than two-fold change in expression during pregnancy compared to baseline levels, with distinct temporal trends through pregnancy. Another 98 genes involved in various biological processes including immune regulation exhibited expression patterns that were differentially associated with pregnancy in the presence or absence of RA. Conclusions Our findings support the hypothesis that the maternal immune system plays an active role during pregnancy, and also provide insight into other systemic changes that occur in the maternal transcriptome during pregnancy compared to the pre-pregnancy state. Only a small proportion of genes modulated by pregnancy were influenced by presence of RA in our data. PMID:26683605

Pregnancy-Induced Changes in Systemic Gene Expression among Healthy Women and Women with Rheumatoid Arthritis.

PubMed

Mittal, Anuradha; Pachter, Lior; Nelson, J Lee; Kjærgaard, Hanne; Smed, Mette Kiel; Gildengorin, Virginia L; Zoffmann, Vibeke; Hetland, Merete Lund; Jewell, Nicholas P; Olsen, Jørn; Jawaheer, Damini

2015-01-01

Pregnancy induces drastic biological changes systemically, and has a beneficial effect on some autoimmune conditions such as rheumatoid arthritis (RA). However, specific systemic changes that occur as a result of pregnancy have not been thoroughly examined in healthy women or women with RA. The goal of this study was to identify genes with expression patterns associated with pregnancy, compared to pre-pregnancy as baseline and determine whether those associations are modified by presence of RA. In our RNA sequencing (RNA-seq) dataset from 5 healthy women and 20 women with RA, normalized expression levels of 4,710 genes were significantly associated with pregnancy status (pre-pregnancy, first, second and third trimesters) over time, irrespective of presence of RA (False Discovery Rate (FDR)-adjusted p value<0.05). These genes were enriched in pathways spanning multiple systems, as would be expected during pregnancy. A subset of these genes (n = 256) showed greater than two-fold change in expression during pregnancy compared to baseline levels, with distinct temporal trends through pregnancy. Another 98 genes involved in various biological processes including immune regulation exhibited expression patterns that were differentially associated with pregnancy in the presence or absence of RA. Our findings support the hypothesis that the maternal immune system plays an active role during pregnancy, and also provide insight into other systemic changes that occur in the maternal transcriptome during pregnancy compared to the pre-pregnancy state. Only a small proportion of genes modulated by pregnancy were influenced by presence of RA in our data.

Interspecies Systems Biology Uncovers Metabolites Affecting C. elegans Gene Expression and Life History Traits

PubMed Central

Watson, Emma; MacNeil, Lesley T.; Ritter, Ashlyn D.; Yilmaz, L. Safak; Rosebrock, Adam P.; Caudy, Amy A.; Walhout, Albertha J. M.

2014-01-01

SUMMARY Diet greatly influences gene expression and physiology. In mammals, elucidating the effects and mechanisms of individual nutrients is challenging due to the complexity of both the animal and its diet. Here we used an interspecies systems biology approach with Caenorhabditis elegans and two if its bacterial diets, Escherichia coli and Comamonas aquatica, to identify metabolites that affect the animal’s gene expression and physiology. We identify vitamin B12 as the major dilutable metabolite provided by Comamonas aq. that regulates gene expression, accelerates development and reduces fertility, but does not affect lifespan. We find that vitamin B12 has a dual role in the animal: it affects development and fertility via the methionine/S-Adenosylmethionine (SAM) cycle and breaks down the short-chain fatty acid propionic acid preventing its toxic buildup. Our interspecies systems biology approach provides a paradigm for understanding complex interactions between diet and physiology. PMID:24529378

Expression pattern of the thrombopoietin receptor (Mpl) in the murine central nervous system.

PubMed

Ivanova, Anna; Wuerfel, Jens; Zhang, Juan; Hoffmann, Olaf; Ballmaier, Matthias; Dame, Christof

2010-07-28

Thrombopoietin (Thpo) and its receptor (Mpl), which regulate megakaryopoiesis, are expressed in the central nervous system (CNS), where Thpo is thought to exert pro-apoptotic effects on newly generated neurons. Mpl expression has been analysed in brain tissue on transcript level and in cultured primary rat neurons and astrocytes on protein level. Herein, we analysed Mpl expression in the developing and adult murine CNS by immunohistochemistry and investigated the brain of mice with homozygous Mpl deficiency (Mpl-/-) by MRI. Mpl was not detectable at developmental stages E12 to E15 in any resident cells of the CNS. From E18 onwards, robust Mpl expression was found in various brain areas, including cerebral cortex, olfactory bulb, thalamus, hypothalamus, medulla, pons, and the grey matter of spinal cord. However, major developmental changes became obvious: In the subventricular zone of the cerebral cortex Mpl expression occurred only during late gestation, while in the hippocampus Mpl expression was detectable for first time at stage P4. In the white matter of the cerebellum Mpl expression was restricted to the perinatal period. In the adult cerebellum, Mpl expression switched to Purkinje cell. The majority of other Mpl-positive cells were NeuN-positive neurons. None of the cells could be double-labelled with astrocyte marker GFAP. Mpl-/- mice showed no gross abnormalities of the brain. Our data locate Mpl expression to neurons at different subdivisions of the spinal cord, rhombencephalon, midbrain and prosencephalon. Besides neuronal cells Mpl protein is also expressed in Purkinje cells of the adult cerebellum.

Development and application of a T7 RNA polymerase-dependent expression system for antibiotic production improvement in Streptomyces.

PubMed

Wei, Junhong; Tian, Jinjin; Pan, Guoqing; Xie, Jie; Bao, Jialing; Zhou, Zeyang

2017-06-01

To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces. A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h. The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.

FGF-2 deficiency does not influence FGF ligand and receptor expression during development of the nigrostriatal system.

PubMed

Ratzka, Andreas; Baron, Olga; Grothe, Claudia

2011-01-01

Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in

Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems.

PubMed

Fischer, Marc J C; Meyer, Sophie; Claudel, Patricia; Perrin, Mireille; Ginglinger, Jean François; Gertz, Claude; Masson, Jean E; Werck-Reinhardt, Danièle; Hugueney, Philippe; Karst, Francis

2013-01-10

Numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (GPP) as a precursor. Sweet basil (Ocimum basilicum) geraniol synthase (GES) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. Grapevine (Vitis vinifera) calli were transformed using Agrobacterium tumefasciens and the plants were regenerated. Thale cress (Arabidopsis thaliana) was transformed using the floral dip method. Tobacco (Nicotiana benthamiana) leaves were agro-infiltrated for transient expression. Although, as expected, geraniol was the main product detected in the leaves, different minor products were observed in these plants (V. vinifera: citronellol and nerol; N. benthamiana: linalool and nerol; A. thaliana: none). O. basilicum GES expression was also carried out with microbial system yeasts (Saccharomyces cerevisiae) and Escherichia coli. These results suggest that the functional properties of a monoterpenol synthase depend not only on the enzyme's amino-acidic sequence, but also on the cellular background. They also suggest that some plant species or microbial expression systems could induce the simultaneous formation of several carbocations, and could thus have a natural tendency to produce a wider spectrum of monoterpenols. Copyright © 2012 Elsevier B.V. All rights reserved.

78 FR 55766 - Submission for Review: Civil Service Retirement System Survivor Annuitant Express Pay Application...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-11

... Annuitant Express Pay Application for Death Benefits, RI 25-51 AGENCY: U.S. Office of Personnel Management... Service Retirement System Survivor Annuitant Express Pay Application for Death Benefits, RI 25-51. As... widower, based on the report of death, when our records show the decedent elected to provide benefits for...

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

PubMed Central

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-01-01

Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice

A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins.

PubMed

Angius, Federica; Ilioaia, Oana; Amrani, Amira; Suisse, Annabelle; Rosset, Lindsay; Legrand, Amélie; Abou-Hamdan, Abbas; Uzan, Marc; Zito, Francesca; Miroux, Bruno

2018-06-05

Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.

Prognostic and predictive values of PD-L1 expression in patients with digestive system cancer: a meta-analysis.

PubMed

Dai, Cong; Wang, Meng; Lu, Jun; Dai, Zhiming; Lin, Shuai; Yang, Pengtao; Tian, Tian; Liu, Xinghan; Min, Weili; Dai, Zhijun

2017-01-01

PD-L1 has been reported to be expressed in diverse human malignancies. However, the prognostic value of PD-L1 in digestive system cancers remains inconclusive. Therefore, we conducted this meta-analysis to evaluate the prognostic impact of PD-L1 expression in digestive system cancers. We searched the PubMed, Embase, and the Chinese National Knowledge Infrastructure for publications concerning PD-L1 expression in digestive system cancers. Correlations of PD-L1 expression level with overall survival (OS), disease-free survival (DFS), and recurrence-free survival (RFS) were analyzed. Finally, 32 studies with 7,308 patients were included. Our results show that PD-L1 expression was significantly associated with poorer OS (hazard ratio [HR] =1.44, 95% confidence interval [CI] =1.18-1.76, P <0.001), but not DFS (HR =0.91, 95% CI =0.61-1.37, P =0.657) or RFS (HR =1.27, 95% CI =0.75-2.14, P =0.368). Moreover, in the subgroup analysis, significant associations between PD-L1 expression and OS were found in Asians (HR =1.50, 95% CI =1.19-1.89, P =0.001), gastric cancer (HR =1.43, 95% CI =1.05-1.94, P =0.021), and pancreatic carcinoma (HR =2.64, 95% CI =1.78-3.93, P <0.001). These results suggest that the expression of PD-L1 is associated with worse OS in digestive system cancers, especially in gastric cancer and pancreatic cancer. In addition, PD-L1 may act as a new parameter for predicting poor prognosis and a promising target for anticancer therapy in digestive system cancers.

A novel gene expression-based prognostic scoring system to predict survival in gastric cancer

DOE PAGES

Wang, Pin; Wang, Yunshan; Hang, Bo; ...

2016-07-11

Analysis of gene expression patterns in gastric cancer (GC) can help to identify a comprehensive panel of gene biomarkers for predicting clinical outcomes and to discover potential new therapeutic targets. Here, a multi-step bioinformatics analytic approach was developed to establish a novel prognostic scoring system for GC. We first identified 276 genes that were robustly differentially expressed between normal and GC tissues, of which, 249 were found to be significantly associated with overall survival (OS) by univariate Cox regression analysis. The biological functions of 249 genes are related to cell cycle, RNA/ncRNA process, acetylation and extracellular matrix organization. A networkmore » was generated for view of the gene expression architecture of 249 genes in 265 GCs. Finally, we applied a canonical discriminant analysis approach to identify a 53-gene signature and a prognostic scoring system was established based on a canonical discriminant function of 53 genes. The prognostic scores strongly predicted patients with GC to have either a poor or good OS. Our study raises the prospect that the practicality of GC patient prognosis can be assessed by this prognostic scoring system.« less

A novel gene expression-based prognostic scoring system to predict survival in gastric cancer

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

Wang, Pin; Wang, Yunshan; Hang, Bo

Analysis of gene expression patterns in gastric cancer (GC) can help to identify a comprehensive panel of gene biomarkers for predicting clinical outcomes and to discover potential new therapeutic targets. Here, a multi-step bioinformatics analytic approach was developed to establish a novel prognostic scoring system for GC. We first identified 276 genes that were robustly differentially expressed between normal and GC tissues, of which, 249 were found to be significantly associated with overall survival (OS) by univariate Cox regression analysis. The biological functions of 249 genes are related to cell cycle, RNA/ncRNA process, acetylation and extracellular matrix organization. A networkmore » was generated for view of the gene expression architecture of 249 genes in 265 GCs. Finally, we applied a canonical discriminant analysis approach to identify a 53-gene signature and a prognostic scoring system was established based on a canonical discriminant function of 53 genes. The prognostic scores strongly predicted patients with GC to have either a poor or good OS. Our study raises the prospect that the practicality of GC patient prognosis can be assessed by this prognostic scoring system.« less

Uncovering the Role of RNA-Binding Proteins in Gene Expression in the Immune System

PubMed Central

Díaz-Muñoz, Manuel D.; Turner, Martin

2018-01-01

Fighting external pathogens requires an ever-changing immune system that relies on tight regulation of gene expression. Transcriptional control is the first step to build efficient responses while preventing immunodeficiencies and autoimmunity. Post-transcriptional regulation of RNA editing, location, stability, and translation are the other key steps for final gene expression, and they are all controlled by RNA-binding proteins (RBPs). Nowadays we have a deep understanding of how transcription factors control the immune system but recent evidences suggest that post-transcriptional regulation by RBPs is equally important for both development and activation of immune responses. Here, we review current knowledge about how post-transcriptional control by RBPs shapes our immune system and discuss the perspective of RBPs being the key players of a hidden immune cell epitranscriptome. PMID:29875770

Uncovering the Role of RNA-Binding Proteins in Gene Expression in the Immune System.

PubMed

Díaz-Muñoz, Manuel D; Turner, Martin

2018-01-01

Fighting external pathogens requires an ever-changing immune system that relies on tight regulation of gene expression. Transcriptional control is the first step to build efficient responses while preventing immunodeficiencies and autoimmunity. Post-transcriptional regulation of RNA editing, location, stability, and translation are the other key steps for final gene expression, and they are all controlled by RNA-binding proteins (RBPs). Nowadays we have a deep understanding of how transcription factors control the immune system but recent evidences suggest that post-transcriptional regulation by RBPs is equally important for both development and activation of immune responses. Here, we review current knowledge about how post-transcriptional control by RBPs shapes our immune system and discuss the perspective of RBPs being the key players of a hidden immune cell epitranscriptome.

Expression of the Wilms' tumor gene WT1 in the murine urogenital system.

PubMed

Pelletier, J; Schalling, M; Buckler, A J; Rogers, A; Haber, D A; Housman, D

1991-08-01

The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression

A Tupaia paramyxovirus vector system for targeting and transgene expression.

PubMed

Engeland, Christine E; Bossow, Sascha; Hudacek, Andrew W; Hoyler, Birgit; Förster, Judith; Veinalde, Rūta; Jäger, Dirk; Cattaneo, Roberto; Ungerechts, Guy; Springfeld, Christoph

2017-09-01

Viruses from the diverse family of Paramyxoviridae include important pathogens and are applied in gene therapy and for cancer treatment. The Tupaia paramyxovirus (TPMV), isolated from the kidney of a tree shrew, does not infect human cells and neutralizing antibodies against other Paramyxoviridae do not cross-react with TPMV. Here, we present a vector system for de novo generation of infectious TPMV that allows for insertion of additional genes as well as targeting using antibody single-chain variable fragments. We show that the recombinant TPMV specifically infect cells expressing the targeted receptor and replicate in human cells. This vector system provides a valuable tool for both basic research and therapeutic applications.

Expression of the mRNAs encoding the limbic system-associated membrane protein (LAMP): II. Fetal rat brain.

PubMed

Pimenta, A F; Reinoso, B S; Levitt, P

1996-11-11

The limbic system-associated membrane protein (LAMP) is a 64-68 kDa neuronal surface glycoprotein expressed in cortical and subcortical regions of the limbic system of the adult and developing rat central nervous system (CNS). LAMP is a member of the immunoglobulin superfamily of cell adhesion molecules with three Ig domains and is highly conserved between rat and human. In this study, the temporal and spatial pattern of lamp gene expression during fetal rat development was analyzed by using Northern blot analysis and in situ hybridization. In Northern blot analysis, two lamp mRNA transcripts, 1.6 kb and 8.0 kb, identical in size to those present in the adult rat nervous system, were detected in developing neural tissue. In situ hybridization analysis showed close correlation, though not identity, between the expression of lamp mRNAs and the distribution of LAMP in limbic regions of the developing rat CNS, indicative of a more complex regulation of gene expression than was previously thought to be the case. The expression of lamp mRNAs is first detected on about embryonic day (E) 13. The hybridization signal is not seen in the proliferative ventricular zone at any level of the neuraxis, indicating that lamp is expressed in postmitotic neurons. In the cerebral cortex, lamp mRNAs are expressed in limbic cortical regions, such as the perirhinal cortex, prefrontal cortex, and cingulate cortex. In the hippocampus, the hybridization signal is observed in Ammon's horn by E18. The neostriatum, amygdaloid complex, and most hypothalamic areas express lamp mRNAs from early stages (E13-E14) in a pattern consistent with the onset of neurogenesis. The emerging patterns of lamp expression at the outset are similar to those seen in adult hypothalamus and dorsal thalamus. Although the hybridization signal is observed in some nonlimbic areas, including midbrain and hindbrain structures, intense labeling is evident in more classic limbic regions. The high levels of expression of lamp

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

PubMed

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

2017-11-21

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

Antimicrobial peptide production and plant-based expression systems for medical and agricultural biotechnology.

PubMed

Holaskova, Edita; Galuszka, Petr; Frebort, Ivo; Oz, M Tufan

2015-11-01

Antimicrobial peptides (AMPs) are vital components of the innate immune system of nearly all living organisms. They generally act in the first line of defense against various pathogenic bacteria, parasites, enveloped viruses and fungi. These low molecular mass peptides are considered prospective therapeutic agents due to their broad-spectrum rapid activity, low cytotoxicity to mammalian cells and unique mode of action which hinders emergence of pathogen resistance. In addition to medical use, AMPs can also be employed for development of innovative approaches for plant protection in agriculture. Conferred disease resistance by AMPs might help us surmount losses in yield, quality and safety of agricultural products due to plant pathogens. Heterologous expression in plant-based systems, also called plant molecular farming, offers cost-effective large-scale production which is regarded as one of the most important factors for clinical or agricultural use of AMPs. This review presents various types of AMPs as well as plant-based platforms ranging from cell suspensions to whole plants employed for peptide production. Although AMP production in plants holds great promises for medicine and agriculture, specific technical limitations regarding product yield, function and stability still remain. Additionally, establishment of particular stable expression systems employing plants or plant tissues generally requires extended time scale for platform development compared to certain other heterologous systems. Therefore, fast and promising tools for evaluation of plant-based expression strategies and assessment of function and stability of the heterologously produced AMPs are critical for molecular farming and plant protection. Copyright © 2015 Elsevier Inc. All rights reserved.

Systems for the expression of orthogonal translation components in eubacterial host cells

DOEpatents

Ryu, Youngha; Schultz, Peter G.

2013-01-22

The invention related to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural acids at genetically-programmed positions.

Systems for the expression of orthogonal translation components in eubacterial host cells

DOEpatents

Ryu, Youngha [San Diego, CA; Schultz, Peter G [La Jolla, CA

2011-06-14

The invention relates to compositions and methods for the in vivo production of polypeptides comprising one or more unnatural amino acids. Specifically, the invention provides plasmid systems for the efficient eubacterial expression of polypeptides comprising one or more unnatural amino acids at genetically-programmed positions.

Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors.

PubMed

Lesch, H P; Laitinen, A; Peixoto, C; Vicente, T; Makkonen, K-E; Laitinen, L; Pikkarainen, J T; Samaranayake, H; Alves, P M; Carrondo, M J T; Ylä-Herttuala, S; Airenne, K J

2011-06-01

Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.

A vesicular glutamate transporter in lampreys: cDNA cloning and early expression in the nervous system.

PubMed

Villar-Cerviño, Verona; Rocancourt, Claire; Menuet, Arnaud; Da Silva, Corinne; Wincker, Patrick; Anadón, Ramón; Mazan, Sylvie; Rodicio, Maria Celina

2010-09-01

Vesicular glutamate transporters (VGLUTs) accumulate glutamate into synaptic vesicles of glutamatergic neurons, and thus are considered to define the phenotype of these neurons. Glutamate also appears to play a role in the development of the nervous system of vertebrates. Here we report the characterization of a vesicular glutamate transporter of lamprey (lVGluT), a novel member of the VGluT gene family. Phylogenetic analysis indicates that lVGLUT cannot be assigned to any of the three VGLUT isoforms characterized in teleosts and mammals, suggesting that these classes may have been fixed after the splitting between cyclostomes and gnathostomes. Expression pattern analysis during lamprey embryogenesis and prolarval stages shows that lVGluT expression is restricted to the nervous system. The first structure to express lVGluT was the olfactory epithelium of late embryos. In the brain of early prolarvae, lVGluT was expressed in most of the neuronal populations that generate the early axonal scaffold. lVGluT expression was also observed in neuronal populations of the rhombencephalon and spinal cord and in ganglia of the branchiomeric, octaval and posterior lateral line nerves. In the rhombencephalon, lVGluT expression appears to be spatially restricted in dorsal and ventral longitudinal domains. Comparison of the early expression of VGluT genes between the lamprey and some anamniotan gnathostomes (frog, zebrafish) reveals a conserved expression pattern, likely to reflect ancestral vertebrate characteristics. 2010 Elsevier B.V. All rights reserved.

GeoMEx: Geographic Information System (GIS) Prototype for Mars Express Data

NASA Astrophysics Data System (ADS)

Manaud, N.; Frigeri, A.; Ivanov, A. B.

2013-09-01

As of today almost a decade of observational data have been returned by the multidisciplinary instruments on-board the ESA's Mars Express spacecraft. All data are archived into the ESA's Planetary Science Archive (PSA), which is the central repository for all ESA's Solar System missions [1]. Data users can perform advanced queries and retrieve data from the PSA using graphical and map-based search interfaces, or via direct FTP download [2]. However the PSA still offers limited geometrical search and visualisation capabilities that are essential for scientists to identify their data of interest. A former study has shown [3] that this limitation is mostly due to the fact that (1) only a subset of the instruments observations geometry information has been modeled and ingested into the PSA, and (2) that the access to that information from GIS software is impossible without going through a cumbersome and undocumented process. With the increasing number of Mars GIS data sets available to the community [4], GIS software have become invaluable tools for researchers to capture, manage, visualise, and analyse data from various sources. Although Mars Express surface imaging data are natural candidates for use in a GIS environment, other non-imaging instruments data (subsurface, atmosphere, plasma) integration is being investigated [5]. The objective of this work is to develop a GIS prototype that will integrate all the Mars Express instruments observations geometry information into a spatial database that can be accessed from external GIS software using standard WMS and WFS protocols. We will firstly focus on the integration of surface and subsurface instruments data (HRSC, OMEGA, MARSIS). In addition to the geometry information, base and context maps of Mars derived from surface mapping instruments data will also be ingested into the system. The system back-end architecture will be implemented using open-source GIS frameworks: PostgreSQL/PostGIS for the database, and Map

Embryo-specific expression of a visual reporter gene as a selection system for citrus transformation

PubMed Central

Zambon, Flavia T.; Erpen, Lígia; Soriano, Leonardo; Grosser, Jude

2018-01-01

The embryo-specific Dc3 gene promoter driving the VvMybA1 anthocyanin regulatory gene was used to develop a visual selection system for the genetic transformation of citrus. Agrobacterium-mediated transformation of cell suspension cultures resulted in the production of purple transgenic somatic embryos that could be easily separated from the green non-transgenic embryos. The somatic embryos produced phenotypically normal plants devoid of any visual purple coloration. These results were also confirmed using protoplast transformation. There was minimal gene expression in unstressed one-year-old transgenic lines. Cold and drought stress did not have any effect on gene expression, while exogenous ABA and NaCl application resulted in a minor change in gene expression in several transgenic lines. When gas exchange was measured in intact leaves, the transgenic lines were similar to controls under the same environment. Our results provide conclusive evidence for the utilization of a plant-derived, embryo-specific visual reporter system for the genetic transformation of citrus. Such a system could aid in the development of an all-plant, consumer-friendly GM citrus tree. PMID:29293649

A novel riboregulator switch system of gene expression for enhanced microbial production of succinic acid.

PubMed

Wang, Jing; Wang, Haoyuan; Yang, Le; Lv, Liping; Zhang, Zhe; Ren, Bin; Dong, Lichun; Li, Ning

2018-04-01

In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between "ON" and "OFF" by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA-RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at "ON" state and that of pepc and ecaA genes were controlled at the "OFF" state in the lag phase and switched to the "OFF" and "ON" state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91 g g -1 and 3.25 g L -1  h -1 , respectively, much higher than those using the strains without harboring the riboregulator switch system.

Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

USGS Publications Warehouse

Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

2001-01-01

This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

ClinData Express – A Metadata Driven Clinical Research Data Management System for Secondary Use of Clinical Data

PubMed Central

Li, Zuofeng; Wen, Jingran; Zhang, Xiaoyan; Wu, Chunxiao; Li, Zuogao; Liu, Lei

2012-01-01

Aim to ease the secondary use of clinical data in clinical research, we introduce a metadata driven web-based clinical data management system named ClinData Express. ClinData Express is made up of two parts: 1) m-designer, a standalone software for metadata definition; 2) a web based data warehouse system for data management. With ClinData Express, what the researchers need to do is to define the metadata and data model in the m-designer. The web interface for data collection and specific database for data storage will be automatically generated. The standards used in the system and the data export modular make sure of the data reuse. The system has been tested on seven disease-data collection in Chinese and one form from dbGap. The flexibility of system makes its great potential usage in clinical research. The system is available at http://code.google.com/p/clindataexpress. PMID:23304327

Engrailed negatively regulates the expression of cell adhesion molecules connectin and neuroglian in embryonic Drosophila nervous system.

PubMed

Siegler, M V; Jia, X X

1999-02-01

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two "effector" genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. We argue that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity.

In vivo imaging of inducible tyrosinase gene expression with an ultrasound array-based photoacoustic system

NASA Astrophysics Data System (ADS)

Harrison, Tyler; Paproski, Robert J.; Zemp, Roger J.

2012-02-01

Tyrosinase, a key enzyme in the production of melanin, has shown promise as a reporter of genetic activity. While green fluorescent protein has been used extensively in this capacity, it is limited in its ability to provide information deep in tissue at a reasonable resolution. As melanin is a strong absorber of light, it is possible to image gene expression using tyrosinase with photoacoustic imaging technologies, resulting in excellent resolutions at multiple-centimeter depths. While our previous work has focused on creating and imaging MCF-7 cells with doxycycline-controlled tyrosinase expression, we have now established the viability of these cells in a murine model. Using an array-based photoacoustic imaging system with 5 MHz center frequency, we capture interleaved ultrasound and photoacoustic images of tyrosinase-expressing MCF-7 tumors both in a tissue mimicking phantom, and in vivo. Images of both the tyrosinase-expressing tumor and a control tumor are presented as both coregistered ultrasound-photoacoustic B-scan images and 3-dimensional photoacoustic volumes created by mechanically scanning the transducer. We find that the tyrosinase-expressing tumor is visible with a signal level 12dB greater than that of the control tumor in vivo. Phantom studies with excised tumors show that the tyrosinase-expressing tumor is visible at depths in excess of 2cm, and have suggested that our imaging system is sensitive to a transfection rate of less than 1%.

A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

PubMed Central

2012-01-01

Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller Sef

[Regulating human interferon-gamma gene expression in marrow stromal cells in mice by Tet-off system].

PubMed

Qin, Xin-Tian; Lu, Yue; Tan, Yin-Duo; Chen, Xiao-Qin; Gen, Qi-Rong

2008-01-01

We have constructed plasmid "pTre-IFN-gamma" and proved that the Tet-off system could regulate the expression of human interferon-gamma (IFN-gamma) gene in murine marrow stromal cells in vitro. This study was to investigate the regulatory reversibility of Tet-off system and its effect on the expression of human IFN-gamma gene in murine marrow stromal cells in mice. Plasmids pTet-off and pTre-IFN-gamma were co-transfected into murine marrow stromal cells. The expression of IFN-gamma in marrow stromal cells was detected with ELISA. The marrow stromal cells were transfused into BABL/c naked mice after co-transfection. The expression of IFN-gamma mRNA in the spleen was detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). IFN-gamma protein was detected in the culture solution of marrow stromal cells after co-transfection. The secretion peak appeared within the first 72 h. The protein level of IFN-gamma was significantly lower in 300 ng/ml tetracycline hydrochloride-treated marrow stroma cells than in untreated cells [(67.11+/-22.14) pg/1 x 10(7) cells vs. (319.96+/-29.04) pg/1 x 10(7) cells, P<0.001]; its expression was increased when removed tetracycline hydrochloride (P=0.032). The expression of human IFN-gamma mRNA was detected in the spleen. The mRNA level of IFN-gamma was significantly higher in untreated group than in continuous tetracycline hydrochloride-treated group [(1.5+/-0.7)x10(5) copies . (100 mg)(-1) vs. (6.9+/-5.3)x10(2) copies . (100 mg)(-1), P<0.001]; its expression in the mice received tetracycline hydrochloride for one single time lay between the above two groups with significant difference. In mice, Tet-off system could rapidly, efficiently and reversibly regulate the expression of human IFN-gamma gene in marrow stromal cells in vitro and in vivo.

An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori

PubMed Central

Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

2015-01-01

We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs. PMID:25739894

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

PubMed Central

Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

2012-01-01

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction. PMID:22093611

Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

PubMed Central

Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

2015-01-01

The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (P<0.05). All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level. PMID:25845365

Expression of and secretion through the Aeromonas salmonicida type III secretion system.

PubMed

Ebanks, Roger O; Knickle, Leah C; Goguen, Michel; Boyd, Jessica M; Pinto, Devanand M; Reith, Michael; Ross, Neil W

2006-05-01

Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 degrees C, but not at its more natural growth temperature of 17 degrees C. More modest increases in expression occur at 24 degrees C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 degrees C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 degrees C in salt concentrations ranging from 0.19 to 0.38 M NaCl. It is also shown that growth at 28 degrees C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.

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.

The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity.

PubMed

Caddell, Daniel F; Wei, Tong; Park, Chang-Jin; Ronald, Pamela C

2015-05-05

Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21 , recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas , and confers robust resistance to X. oryzae pv. oryzae ( Xoo ). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21 . Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression.

The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity

PubMed Central

Caddell, Daniel F.; Wei, Tong; Park, Chang-Jin; Ronald, Pamela C.

2016-01-01

Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression. PMID:27525297

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

PubMed

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

Dissociation of sad facial expressions and autonomic nervous system responding in boys with disruptive behavior disorders

PubMed Central

Marsh, Penny; Beauchaine, Theodore P.; Williams, Bailey

2009-01-01

Although deficiencies in emotional responding have been linked to externalizing behaviors in children, little is known about how discrete response systems (e.g., expressive, physiological) are coordinated during emotional challenge among these youth. We examined time-linked correspondence of sad facial expressions and autonomic reactivity during an empathy-eliciting task among boys with disruptive behavior disorders (n = 31) and controls (n = 23). For controls, sad facial expressions were associated with reduced sympathetic (lower skin conductance level, lengthened cardiac preejection period [PEP]) and increased parasympathetic (higher respiratory sinus arrhythmia [RSA]) activity. In contrast, no correspondence between facial expressions and autonomic reactivity was observed among boys with conduct problems. Furthermore, low correspondence between facial expressions and PEP predicted externalizing symptom severity, whereas low correspondence between facial expressions and RSA predicted internalizing symptom severity. PMID:17868261

Expressive facial animation synthesis by learning speech coarticulation and expression spaces.

PubMed

Deng, Zhigang; Neumann, Ulrich; Lewis, J P; Kim, Tae-Yong; Bulut, Murtaza; Narayanan, Shrikanth

2006-01-01

Synthesizing expressive facial animation is a very challenging topic within the graphics community. In this paper, we present an expressive facial animation synthesis system enabled by automated learning from facial motion capture data. Accurate 3D motions of the markers on the face of a human subject are captured while he/she recites a predesigned corpus, with specific spoken and visual expressions. We present a novel motion capture mining technique that "learns" speech coarticulation models for diphones and triphones from the recorded data. A Phoneme-Independent Expression Eigenspace (PIEES) that encloses the dynamic expression signals is constructed by motion signal processing (phoneme-based time-warping and subtraction) and Principal Component Analysis (PCA) reduction. New expressive facial animations are synthesized as follows: First, the learned coarticulation models are concatenated to synthesize neutral visual speech according to novel speech input, then a texture-synthesis-based approach is used to generate a novel dynamic expression signal from the PIEES model, and finally the synthesized expression signal is blended with the synthesized neutral visual speech to create the final expressive facial animation. Our experiments demonstrate that the system can effectively synthesize realistic expressive facial animation.

Techno-economic analysis of horseradish peroxidase production using a transient expression system in Nicotiana benthamiana.

PubMed

Walwyn, David Richard; Huddy, Suzanne M; Rybicki, Edward P

2015-01-01

Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

Absolute gene expression patterns of thioredoxin and glutaredoxin redox systems in mouse.

PubMed

Jurado, Juan; Prieto-Alamo, María-José; Madrid-Rísquez, José; Pueyo, Carmen

2003-11-14

This work provides the first absolute expression patterns of genes coding for all known components of both thioredoxin (Trx) and glutaredoxin (Grx) systems in mouse: Trx1, Trx2, Grx1, Grx2, TrxR1, TrxR2, thioredoxin/glutathione reductase, and glutathione reductase. We devised a novel assay that, combining the advantages of multiplex and real-time PCR, streamlines the quantitation of the actual mRNA copy numbers in whole-animal experiments. Quantitations reported establish differences among adult organs and embryonic stages, compare mRNA decay rates, explore the significance of alternative mRNA isoforms derived from TrxR1 and Grx2 genes, and examine the time-course expression upon superoxide stress promoted by paraquat. Collectively, these quantitations show: i) unique expression profiles for each transcript and mouse organ examined, yet with some general trends like the higher amounts of mRNA species coding for thioredoxins than those coding for the reductases that control their redox states and activities; ii) continuous expression during embryogenesis with outstanding up-regulations of Trx1 and TrxR1 mRNAs in specific temporal sequences; iii) drastic differences in mRNA stability, liver decay rates range from 2.8 h (thioredoxin/glutathione reductase) to >/= 35 h (Trx1 and Trx2), and directly correlate with mRNA steady-state values; iv) testis-specific differences in the amounts (relative to total isoforms) of transcripts yielding the mitochondrial Grx2a and 67-kDa TrxR1 variants; and v) coordinated up-regulation of TrxR1 and glutathione reductase mRNAs in response to superoxide stress in an organ-specific manner. Further insights into in vivo roles of these redox systems should be gained from more focused studies of the mechanisms underlying the vast differences reported here at the transcript level.

The leptin system and its expression at different nutritional and pregnant stages in lined seahorse (Hippocampus erectus).

PubMed

Zhang, Huixian; Qin, Geng; Zhang, Yanhong; Li, Shuisheng; Lin, Qiang

2016-10-15

Leptin is an essential hormone for the regulation of energy metabolism and food intake in vertebrate animals. To better understand the physiological roles of leptin in nutrient regulation in paternal ovoviviparous fish (family Syngnathidae), the present study cloned the full-length of leptin-a and leptin receptor (lepr) genes in lined seahorse (Hippocampus erectus). Results showed that there was a 576-bp intron between two exons in leptin-a gene but no leptin-b gene in seahorse. Although the primary amino acid sequence conservation of seahorse leptin-a was very low, the 3-D structure modeling of seahorse leptin-a revealed strong conservation of tertiary structure with other vertebrates. Seahorse leptin-a mRNA was highly expressed in brain, whereas lepr mRNA was mainly expressed in ovary and gill. Interestingly, both leptin-a and lepr mRNA were expressed in the brood pouch of male seahorse, suggesting the leptin system plays a role during the male pregnancy. Physiological experiments showed that the expression of hepatic leptin-a and lepr mRNA in unfed seahorses was significantly higher than that in those fed 100%, as well as 60%, of their food during the fasting stage, showing that seahorse might initiate the leptin system to regulate its energy metabolism while starving. Moreover, the expression of leptin-a in the brood pouch of pregnant seahorse was significantly upregulated compared with non-pregnant seahorse, whereas the expression of lepr was downregulated, suggesting that the leptin system might be involved in the male pregnancy. In conclusion, the leptin system plays a role in the energy metabolism and food intake, and might provide new insights into molecular regulation of male pregnancy in seahorse. © 2016. Published by The Company of Biologists Ltd.

The leptin system and its expression at different nutritional and pregnant stages in lined seahorse (Hippocampus erectus)

PubMed Central

Zhang, Huixian; Qin, Geng; Zhang, Yanhong; Li, Shuisheng

2016-01-01

ABSTRACT Leptin is an essential hormone for the regulation of energy metabolism and food intake in vertebrate animals. To better understand the physiological roles of leptin in nutrient regulation in paternal ovoviviparous fish (family Syngnathidae), the present study cloned the full-length of leptin-a and leptin receptor (lepr) genes in lined seahorse (Hippocampus erectus). Results showed that there was a 576-bp intron between two exons in leptin-a gene but no leptin-b gene in seahorse. Although the primary amino acid sequence conservation of seahorse leptin-a was very low, the 3-D structure modeling of seahorse leptin-a revealed strong conservation of tertiary structure with other vertebrates. Seahorse leptin-a mRNA was highly expressed in brain, whereas lepr mRNA was mainly expressed in ovary and gill. Interestingly, both leptin-a and lepr mRNA were expressed in the brood pouch of male seahorse, suggesting the leptin system plays a role during the male pregnancy. Physiological experiments showed that the expression of hepatic leptin-a and lepr mRNA in unfed seahorses was significantly higher than that in those fed 100%, as well as 60%, of their food during the fasting stage, showing that seahorse might initiate the leptin system to regulate its energy metabolism while starving. Moreover, the expression of leptin-a in the brood pouch of pregnant seahorse was significantly upregulated compared with non-pregnant seahorse, whereas the expression of lepr was downregulated, suggesting that the leptin system might be involved in the male pregnancy. In conclusion, the leptin system plays a role in the energy metabolism and food intake, and might provide new insights into molecular regulation of male pregnancy in seahorse. PMID:27628034

Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System

PubMed Central

Shinzawa, Naoaki; Nishikawa, Sayaka; Suzuki, Koichiro; Fukui-Miyazaki, Aya

2018-01-01

ABSTRACT We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica, which is required for O antigen biosynthesis, into the chromosome of B. pertussis, which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti-B. bronchiseptica antibody but not the anti-B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica. O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis, which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant

Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System.

PubMed

Ishigaki, Keisuke; Shinzawa, Naoaki; Nishikawa, Sayaka; Suzuki, Koichiro; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko

2018-01-01

We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica , which is required for O antigen biosynthesis, into the chromosome of B. pertussis , which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti- B. bronchiseptica antibody but not the anti- B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica . O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis , which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant

Developmental expression and distribution of nesfatin-1/NUCB2 in the canine digestive system.

PubMed

Jiang, Shudong; Zhou, Weijuan; Zhang, Xingwang; Wang, Dengfeng; Zhu, Hui; Hong, Meizhen; Gong, Yajing; Ye, Jing; Fang, Fugui

2016-03-01

Nesfatin-1/NUCB2 is a neuropeptide that plays important roles in regulating food intake and energy homeostasis. The distribution of nesfatin-1/NUCB2 protein and mRNA has not been investigated in the canine digestive system. The present study was conducted to evaluate the expression of nesfatin-1/NUCB2 protein and NUCB2 mRNA in the canine digestive organs (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver and pancreas). The tissues of the digestive system were collected from dogs at different developmental stages (infantile, juvenile, pubertal and adult). Nesfatin-1/NUCB2 protein localization in the organs of adult dogs was detected by immunohistochemistry. The expression of NUCB2 mRNA at the four developmental stages was analyzed by real-time fluorescence quantitative PCR (qRT-PCR). Nesfatin-1/NUCB2 protein was distributed in the fundic gland region of the stomach, and the islet area and exocrine portions of the pancreas. However, NUCB2 mRNA was found in all digestive organs, although the expression levels in the pancreas and stomach were higher than those in liver, duodenum and other digestive tract tissues (P<0.05) at the four different developmental stages of the dogs. In this study, nesfatin-1/NUCB2 was found to be present at high levels in the stomach and pancreas at both the protein and mRNA levels; however, NUCB2 expression was found at lower levels in all of the digestive organs. These findings provide the basis of further investigations to elucidate the functions of nefatin-1 in the canine digestive system. Copyright © 2015 Elsevier GmbH. All rights reserved.

Robust patterning of gene expression based on internal coordinate system of cells.

PubMed

Ogawa, Ken-ichiro; Miyake, Yoshihiro

2015-06-01

Cell-to-cell communication in multicellular organisms is established through the transmission of various kinds of chemical substances such as proteins. It is well known that gene expression triggered by a chemical substance in individuals has stable spatial patterns despite the individual differences in concentration patterns of the chemical substance. This fact reveals an important property of multicellular organisms called "robustness", which allows the organisms to generate their forms while maintaining proportion. Robustness has been conventionally accounted for by the stability of solutions of dynamical equations that represent a specific interaction network of chemical substances. However, any biological system is composed of autonomous elements. In general, an autonomous element does not merely accept information on the chemical substance from the environment; instead, it accepts the information based on its own criteria for reaction. Therefore, this phenomenon needs to be considered from the viewpoint of cells. Such a viewpoint is expected to allow the consideration of the autonomy of cells in multicellular organisms. This study aims to explain theoretically the robust patterning of gene expression from the viewpoint of cells. For this purpose, we introduced a new operator for transforming a state variable of a chemical substance from an external coordinate system to an internal coordinate system of each cell, which describes the observation of the chemical substance by cells. We then applied this operator to the simplest reaction-diffusion model of the chemical substance to investigate observation effects by cells. Our mathematical analysis of this extended model indicates that the robust patterning of gene expression against individual differences in concentration pattern of the chemical substance can be explained from the viewpoint of cells if there is a regulation field that compensates for the difference between cells seen in the observation results

Systems biology definition of the core proteome of metabolism and expression is consistent with high-throughput data.

PubMed

Yang, Laurence; Tan, Justin; O'Brien, Edward J; Monk, Jonathan M; Kim, Donghyuk; Li, Howard J; Charusanti, Pep; Ebrahim, Ali; Lloyd, Colton J; Yurkovich, James T; Du, Bin; Dräger, Andreas; Thomas, Alex; Sun, Yuekai; Saunders, Michael A; Palsson, Bernhard O

2015-08-25

Finding the minimal set of gene functions needed to sustain life is of both fundamental and practical importance. Minimal gene lists have been proposed by using comparative genomics-based core proteome definitions. A definition of a core proteome that is supported by empirical data, is understood at the systems-level, and provides a basis for computing essential cell functions is lacking. Here, we use a systems biology-based genome-scale model of metabolism and expression to define a functional core proteome consisting of 356 gene products, accounting for 44% of the Escherichia coli proteome by mass based on proteomics data. This systems biology core proteome includes 212 genes not found in previous comparative genomics-based core proteome definitions, accounts for 65% of known essential genes in E. coli, and has 78% gene function overlap with minimal genomes (Buchnera aphidicola and Mycoplasma genitalium). Based on transcriptomics data across environmental and genetic backgrounds, the systems biology core proteome is significantly enriched in nondifferentially expressed genes and depleted in differentially expressed genes. Compared with the noncore, core gene expression levels are also similar across genetic backgrounds (two times higher Spearman rank correlation) and exhibit significantly more complex transcriptional and posttranscriptional regulatory features (40% more transcription start sites per gene, 22% longer 5'UTR). Thus, genome-scale systems biology approaches rigorously identify a functional core proteome needed to support growth. This framework, validated by using high-throughput datasets, facilitates a mechanistic understanding of systems-level core proteome function through in silico models; it de facto defines a paleome.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

PubMed

López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

PubMed Central

Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

Identification of an elaborate NK-specific system regulating HLA-C expression

PubMed Central

Ivarsson, Martin A.; Walker-Sperling, Victoria E.; Subleski, Jeff; Johnson, Jenna K.; Wright, Paul W.; Carrington, Mary; McVicar, Daniel W.

2018-01-01

The HLA-C gene appears to have evolved in higher primates to serve as a dominant source of ligands for the KIR2D family of inhibitory MHC class I receptors. The expression of NK cell-intrinsic MHC class I has been shown to regulate the murine Ly49 family of MHC class I receptors due to the interaction of these receptors with NK cell MHC in cis. However, cis interactions have not been demonstrated for the human KIR and HLA proteins. We report the discovery of an elaborate NK cell-specific system regulating HLA-C expression, indicating an important role for HLA-C in the development and function of NK cells. A large array of alternative transcripts with differences in intron/exon content are generated from an upstream NK-specific HLA-C promoter, and exon content varies between HLA-C alleles due to SNPs in splice donor/acceptor sites. Skipping of the first coding exon of HLA-C generates a subset of untranslatable mRNAs, and the proportion of untranslatable HLA-C mRNA decreases as NK cells mature, correlating with increased protein expression by mature NK cells. Polymorphism in a key Ets-binding site of the NK promoter has generated HLA-C alleles that lack significant promoter activity, resulting in reduced HLA-C expression and increased functional activity. The NK-intrinsic regulation of HLA-C thus represents a novel mechanism controlling the lytic activity of NK cells during development. PMID:29329284

Expression of group III metabotropic glutamate receptors in the reproductive system of male mice.

PubMed

Marciniak, Marcin; Chruścicka, Barbara; Lech, Tomasz; Burnat, Grzegorz; Pilc, Andrzej

2016-03-01

Although the presence of metabotropic glutamate (mGlu) receptors in the central nervous system is well documented, they have recently been found in peripheral and non-neuronal tissues. In the present study we investigated the expression of group III mGlu receptors in the reproductive system of male mice. Reverse transcription-polymerase chain reaction analysis revealed the presence of mGlu6, mGlu7 and mGlu8 (but not mGlu4) receptor transcripts in testes and epididymides from adult mice. In addition, expression of mGlu6 (Grm6) and mGlu8 receptor (Grm8) mRNA was detected in spermatozoa isolated from the vas deferens. The vas deferens was found to contain only mGlu7 receptor (Grm7) mRNA, which was particularly intense in 21-day-old male mice. In penile homogenates, only the mGlu7 receptor signal was detected. Genetic ablation of the mGlu7 receptor in males led to fertility disorders manifested by decreased insemination capability as well as deterioration of sperm parameters, particularly sperm motility, vitality, sperm membrane integrity and morphology, with a simultaneous increase in sperm concentration. These results indicate that constitutively expressed mGlu receptors in the male reproductive system may play an important role in ejaculation and/or erection processes, as well as in the formation and maturation of spermatozoa.

Altered expression of IGF-I system in neurons of the inflamed spinal cord during acute experimental autoimmune encephalomyelitis.

PubMed

Parvaneh Tafreshi, Azita; Talebi, Farideh; Ghorbani, Samira; Bernard, Claude; Noorbakhsh, Farshid

2017-10-01

There is growing evidence that the impaired IGF-I system contributes to neurodegeneration. In this study, we examined the spinal cords of the EAE, the animal model of multiple sclerosis, to see if the expression of the IGF-I system is altered. To induce EAE, C57/BL6 mice were immunized with the Hooke lab MOG kit, sacrificed at the peak of the disease and their spinal cords were examined for the immunoreactivities (ir) of the IGF-I, IGF binding protein-1 (IGFBP-1) and glycogen synthase kinase 3β (GSK3β), as one major downstream molecule in the IGF-I signaling. Although neurons in the non EAE spinal cords did not show the IGF-I immunoreactivity, they were numerously positive for the IGFBP-1. In the inflamed EAE spinal cord however, the patterns of expressions were reversed, that is, a significant increased number of IGF-I expressing neurons versus a reduced number of IGFBP-1 positive neurons. Moreover, while nearly all IGF-I-ir neurons expressed GSK3β, some expressed it more intensely. Considering our previous finding where we showed a significant reduced number of the inactive (phosphorylated) but not that of the total GSK3β expressing neurons in the EAE spinal cord, it is conceivable that the intense total GSK3β expression in the IGF-I-ir neurons belongs to the active form of GSK3β known to exert neuroinflammatory effects. We therefore suggest that the altered expression of the IGF-I system including GSK3β in spinal cord neurons might involve in pathophysiological events during the EAE. © 2017 Wiley Periodicals, Inc.

Evaluation of the Expressiveness of an ICNP-based Nursing Data Dictionary in a Computerized Nursing Record System

PubMed Central

Cho, InSook; Park, Hyeoun-Ae

2006-01-01

This study evaluated the domain completeness and expressiveness issues of the International Classification for Nursing Practice-based (ICNP) nursing data dictionary (NDD) through its application in an enterprise electronic medical record (EMR) system as a standard vocabulary at a single tertiary hospital in Korea. Data from 2,262 inpatients obtained over a period of 9 weeks (May to July 2003) were extracted from the EMR system for analysis. Among the 530,218 data-input events, 401,190 (75.7%) were entered from the NDD, 20,550 (3.9%) used only free text, and 108,478 (20.4%) used a combination of coded data and free text. A content analysis of the free-text events showed that 80.3% of the expressions could be found in the NDD, whereas 10.9% were context-specific expressions such as direct quotations of patient complaints and responses, and references to the care plan or orders of physicians. A total of 7.8% of the expressions was used for a supplementary purpose such as adding a conjunction or end verb to make an expression appear as natural language. Only 1.0% of the expressions were identified as not being covered by the NDD. This evaluation study demonstrates that the ICNP-based NDD has sufficient power to cover most of the expressions used in a clinical nursing setting. PMID:16622170

Central Nervous System and Vertebrae Development in Horses: a Chronological Study with Differential Temporal Expression of Nestin and GFAP.

PubMed

Rigoglio, Nathia N; Barreto, Rodrigo S N; Favaron, Phelipe O; Jacob, Júlio C F; Smith, Lawrence C; Gastal, Melba O; Gastal, Eduardo L; Miglino, Maria Angélica

2017-01-01

The neural system is one of the earliest systems to develop and the last to be fully developed after birth. This study presents a detailed description of organogenesis of the central nervous system (CNS) at equine embryonic/fetal development between 19 and 115 days of pregnancy. The expression of two important biomarkers in the main structure of the nervous system responsible for neurogenesis in the adult individual, and in the choroid plexus, was demonstrated by Nestin and glial fibrillary acid protein (GFAP) co-labeling. In the 29th day of pregnancy in the undifferentiated lateral ventricle wall, the presence of many cells expressing Nestin and few expressing GFAP was observed. After the differentiation of the lateral ventricle wall zones at 60 days of pregnancy, the subventricular zone, which initially had greater number of Nestin + cells, began to show higher numbers of GFAP + cells at 90 days of pregnancy. A similar pattern was observed for Nestin + and GFAP + cells during development of the choroid plexus. This study demonstrates, for the first time, detailed chronological aspects of the equine central nervous system organogenesis associated with downregulation of Nestin and upregulation of GFAP expression.

Integration Method of Emphatic Motions and Adverbial Expressions with Scalar Parameters for Robotic Motion Coaching System

NASA Astrophysics Data System (ADS)

Okuno, Keisuke; Inamura, Tetsunari

A robotic coaching system can improve humans' learning performance of motions by intelligent usage of emphatic motions and adverbial expressions according to user reactions. In robotics, however, method to control both the motions and the expressions and how to bind them had not been adequately discussed from an engineering point of view. In this paper, we propose a method for controlling and binding emphatic motions and adverbial expressions by using two scalar parameters in a phase space. In the phase space, variety of motion patterns and verbal expressions are connected and can be expressed as static points. We show the feasibility of the proposing method through experiments of actual sport coaching tasks for beginners. From the results of participants' improvements in motion learning, we confirmed the feasibility of the methods to control and bind emphatic motions and adverbial expressions, as well as confirmed contribution of the emphatic motions and positive correlation of adverbial expressions for participants' improvements in motion learning. Based on the results, we introduce a hypothesis that individually optimized method for binding adverbial expression is required.

Connexin32 expression in central and peripheral nervous systems

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

Deschenes, S.M.; Scherer, S.S.; Fischbeck, K.H.

1994-09-01

Mutations have been identified in the gap junction gene, connexin32 (Cx32), in patients affected with the X-linked form of the demyelinating neuropathy, Charcot-Marie-Tooth disease (CMTX). Gap junctions composed of Cx32 are present and developmentally regulated in a wide variety of tissues. In peripheral nerve, our immunohistochemical analysis localized Cx32 to the noncompacted myelin of the paranodal regions and the Schmidt-Lantermann incisures, where previous studies describe gap junctions. In contrast to the location of Cx32 in peripheral nerve and the usual restriction of clinical manifestations to the peripheral nervous system (PNS) (abstract by Paulson describes an exception), preliminary studies show thatmore » Cx32 is present in the compacted myelin of the central nervous system (CNS), as demonstrated by radial staining through the myelin sheath of oligodendrocytes in rat spinal cord. Analysis of Cx32 expression in various regions of rat CNS during development shows that the amount of Cx32 mRNA and protein increases as myelination increases, a pattern observed for other myelin genes. Studies in the PNS provide additional evidence that Cx32 and myelin genes are coordinately regulated at the transcriptional level; Cx32 and peripheral myelin gene PMP-22 mRNAs are expressed in parallel following transient or permanent nerve injury. Differences in post-translational regulation of Cx32 in the CNS and PNS may be indicated by the presence of a faster migrating form of Cs32 in cerebrum versus peripheral nerve. Studies are currently underway to determine the unique role of Cx32 in peripheral nerve.« less

Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

PubMed

Ito, Akira; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Aoyama, Tomoki; Kuroki, Hiroshi

2015-01-01

Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis.

EXPRESS Rack Overview

NASA Technical Reports Server (NTRS)

Sledd, Annette M.; Mueller, Charles W.

1999-01-01

The EXpedite the PRocessing of Experiments to Space Station or EXPRESS Rack System, was developed to provide Space Station accommodations for small, subrack payloads. The EXPRESS Rack accepts Space Shuttle middeck locker type payloads and International Subrack Interface Standard (ISIS) Drawer payloads, allowing previously flown payloads an opportunity to transition to the International Space Station. The EXPRESS Rack provides power, data, command and control, video, water cooling, air cooling, vacuum exhaust, and Nitrogen supply to payloads. The EXPRESS Rack system also includes transportation racks to transport payloads to and from the Space Station, Suitcase Simulators to allow a payload developer to verify power and data interfaces at the development site, Functional Checkout Units to allow Payload checkout at KSC prior to launch, and trainer racks for the astronauts to learn how to operate the EXPRESS Racks prior to flight. Standard hardware and software interfaces provided by the EXPRESS Rack simplify the analytical and physical integration processes, and facilitates simpler ISS payload development. The EXPRESS Rack has also formed the basis for the U.S. Life Sciences payload racks on Space Station.

21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

Code of Federal Regulations, 2010 CFR

2010-04-01

... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control is...

21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

Code of Federal Regulations, 2011 CFR

2011-04-01

... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control is...

21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

Code of Federal Regulations, 2013 CFR

2013-04-01

... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control is...

21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

Code of Federal Regulations, 2012 CFR

2012-04-01

... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control is...

21 CFR 866.6040 - Gene expression profiling test system for breast cancer prognosis.

Code of Federal Regulations, 2014 CFR

2014-04-01

... cancer prognosis. 866.6040 Section 866.6040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... cancer prognosis. (a) Identification. A gene expression profiling test system for breast cancer prognosis... previously diagnosed breast cancer. (b) Classification. Class II (special controls). The special control is...

Programmed Cell Death Ligand 1 Expression in Primary Central Nervous System Lymphomas: A Clinicopathological Study.

PubMed

Hayano, Azusa; Komohara, Yoshihiro; Takashima, Yasuo; Takeya, Hiroto; Homma, Jumpei; Fukai, Junya; Iwadate, Yasuo; Kajiwara, Koji; Ishizawa, Shin; Hondoh, Hiroaki; Yamanaka, Ryuya

2017-10-01

Programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) have been shown to predict response to PD-L1/PD-1-targeted therapy. We analyzed PD-L1 expression in primary central nervous system lymphomas (PCNSLs). PD-L1 protein and mRNA expression were evaluated in 64 PCNSL tissue samples. IFN-γ, IL-10, CD4, and CD8 mRNA expression was also evaluated. PD-L1 protein was detected in tumor cells in 2 (4.1%) cases and in tumor microenvironments in 25 (52%) cases. PD-L1 mRNA positively correlated with IFN-γ (p=0.0024) and CD4 (p=0.0005) mRNA expression. IFN-γ mRNA positively correlated with CD8 mRNA expression (p=0.0001). Furthermore, tumor cell PD-L1 expression correlated positively with overall survival (p=0.0177), whereas microenvironmental PD-L1 expression exhibited an insignificant negative trend with overall survival (p=0.188). PD-L1 was expressed on both tumor and/or tumor-infiltrating immune cells in PCNSL. The biological roles of this marker warrant further investigation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A family of splice variants of CstF-64 expressed in vertebrate nervous systems

PubMed Central

Shankarling, Ganesh S; Coates, Penelope W; Dass, Brinda; MacDonald, Clinton C

2009-01-01

Background Alternative splicing and polyadenylation are important mechanisms for creating the proteomic diversity necessary for the nervous system to fulfill its specialized functions. The contribution of alternative splicing to proteomic diversity in the nervous system has been well documented, whereas the role of alternative polyadenylation in this process is less well understood. Since the CstF-64 polyadenylation protein is known to be an important regulator of tissue-specific polyadenylation, we examined its expression in brain and other organs. Results We discovered several closely related splice variants of CstF-64 – collectively called βCstF-64 – that could potentially contribute to proteomic diversity in the nervous system. The βCstF-64 splice variants are found predominantly in the brains of several vertebrate species including mice and humans. The major βCstF-64 variant mRNA is generated by inclusion of two alternate exons (that we call exons 8.1 and 8.2) found between exons 8 and 9 of the CstF-64 gene, and contains an additional 147 nucleotides, encoding 49 additional amino acids. Some variants of βCstF-64 contain only the first alternate exon (exon 8.1) while other variants contain both alternate exons (8.1 and 8.2). In mice, the predominant form of βCstF-64 also contains a deletion of 78 nucleotides from exon 9, although that variant is not seen in any other species examined, including rats. Immunoblot and 2D-PAGE analyses of mouse nuclear extracts indicate that a protein corresponding to βCstF-64 is expressed in brain at approximately equal levels to CstF-64. Since βCstF-64 splice variant family members were found in the brains of all vertebrate species examined (including turtles and fish), this suggests that βCstF-64 has an evolutionarily conserved function in these animals. βCstF-64 was present in both pre- and post-natal mice and in different regions of the nervous system, suggesting an important role for βCstF-64 in neural gene

Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems.

PubMed

Nishino, Kunihiko

2018-01-01

Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.

Solid-phase fermentation and juice expression systems for sweet sorghum

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

Bryan, W.L.; Monroe, G.E.; Caussariel, P.M.

1985-01-01

Two systems to recover fermented juice from variety M 81E sweet sorghum stalks that contained about 11% fermentable sugar were compared. (a) Stalks with leaves and tops removed were chopped and inoculated with 0.2% yeast in a forage harvester, stored under anaerobic conditions for 75 hours in insulated fermentors and pressed in a screw press to recover fermented juice (5-6% ethanol). (b) Mechanically harvested sweet sorghum billets (30 cm length) without leaves or seed heads were shredded and milled in a 3-roll mill; and bagasse was inoculated with 0.2% yeast, fermented for 100 h and pressed to recover fermented juicemore » (4 to 5% ethanol). Potential ethanol yields were 75% of theoretical for the forage harvest system and 78% for the shredder mill system, based on 95% of theoretical ethanol yield from juice expressed during milling and no loss of ethanol during fermentation, handling and pressing in the screw press. 20 references.« less

New Fluorescent Reporter Systems for Evaluation of the Expression of E- and N-Cadherins.

PubMed

Burmistrova, O A; Nikulin, S V; Zakharova, G S; Fomicheva, K A; Alekseev, B Ya; Shkurnikov, M Yu

2018-05-24

During metastatic growth, cells of solid tumors undergo phenotypical changes related to epithelial-mesenchymal transition. Epithelial-mesenchymal transition is regarded as a potential target for prospective antitumor drugs. Fluorescent reporter systems for evaluation of the expression of markers of epithelial and mesenchymal status (E- and N-cadherins) were created. The described approaches can be used for creation of analogous reporter systems.

Exploration at the Edge of the Solar System: The Pluto-Kuiper Express Mission (Invited)

NASA Astrophysics Data System (ADS)

Terrile, R. J.

1999-09-01

The Pluto-Kuiper Express mission is one component of the Outer Planets/Solar Probe Project which is part of the exploration strategy laid out in the Solar System Exploration Roadmap. The first three missions of this project are the Europa Orbiter, Pluto-Kuiper Express and the Solar Probe. All require challenging new technologies and the ability to operate in deep space and at Jupiter. Use of common management and design approaches, avionics, and mission software is planned to reduce the costs of the three missions. The Pluto-Kuiper Express mission is planned to launch in 2004 and is designed to provide the first reconnaissance of the Solar System's most distant planet, Pluto, and it, moon Charon. A gravity assist from Jupiter will allow an 8-year flight time to Pluto and the possibility of encountering one or more Edgeworth-Kuiper Belt objects after the Pluto encounter. The primary science objectives for the mission include characterizing the global geology and geomorphology of Pluto and Charon, mapping their surface composition and characterizing Pluto's neutral atmosphere and its escape rate. This mission is currently soliciting scientific investigations through a NASA Announcement of Opportunity.

Effect of long-term actual spaceflight on the expression of key genes encoding serotonin and dopamine system

NASA Astrophysics Data System (ADS)

Popova, Nina; Shenkman, Boris; Naumenko, Vladimir; Kulikov, Alexander; Kondaurova, Elena; Tsybko, Anton; Kulikova, Elisabeth; Krasnov, I. B.; Bazhenova, Ekaterina; Sinyakova, Nadezhda

The effect of long-term spaceflight on the central nervous system represents important but yet undeveloped problem. The aim of our work was to study the effect of 30-days spaceflight of mice on Russian biosatellite BION-M1 on the expression in the brain regions of key genes of a) serotonin (5-HT) system (main enzymes in 5-HT metabolism - tryptophan hydroxylase-2 (TPH-2), monoamine oxydase A (MAO A), 5-HT1A, 5-HT2A and 5-HT3 receptors); b) pivotal enzymes in DA metabolism (tyrosine hydroxylase, COMT, MAO A, MAO B) and D1, D2 receptors. Decreased expression of genes encoding the 5-HT catabolism (MAO A) and 5-HT2A receptor in some brain regions was shown. There were no differences between “spaceflight” and control mice in the expression of TPH-2 and 5-HT1A, 5-HT3 receptor genes. Significant changes were found in genetic control of DA system. Long-term spaceflight decreased the expression of genes encoding the enzyme in DA synthesis (tyrosine hydroxylase in s.nigra), DA metabolism (MAO B in the midbrain and COMT in the striatum), and D1 receptor in hypothalamus. These data suggested that 1) microgravity affected genetic control of 5-HT and especially the nigrostriatal DA system implicated in the central regulation of muscular tonus and movement, 2) the decrease in the expression of genes encoding key enzyme in DA synthesis, DA degradation and D1 receptor contributes to the movement impairment and dyskinesia produced by the spaceflight. The study was supported by Russian Foundation for Basic Research grant No. 14-04-00173.

Genetic variations in the dopamine system and facial expression recognition in healthy chinese college students.

PubMed

Zhu, Bi; Chen, Chuansheng; Moyzis, Robert K; Dong, Qi; Chen, Chunhui; He, Qinghua; Stern, Hal S; Li, He; Li, Jin; Li, Jun; Lessard, Jared; Lin, Chongde

2012-01-01

This study investigated the relation between genetic variations in the dopamine system and facial expression recognition. A sample of Chinese college students (n = 478) was given a facial expression recognition task. Subjects were genotyped for 98 loci [96 single-nucleotide polymorphisms (SNPs) and 2 variable number tandem repeats] in 16 genes involved in the dopamine neurotransmitter system, including its 4 subsystems: synthesis (TH, DDC, and DBH), degradation/transport (COMT,MAOA,MAOB, and SLC6A3), receptors (DRD1,DRD2,DRD3,DRD4, and DRD5), and modulation (NTS,NTSR1,NTSR2, and NLN). To quantify the total contributions of the dopamine system to emotion recognition, we used a series of multiple regression models. Permutation analyses were performed to assess the posterior probabilities of obtaining such results. Among the 78 loci that were included in the final analyses (after excluding 12 SNPs that were in high linkage disequilibrium and 8 that were not in Hardy-Weinberg equilibrium), 1 (for fear), 3 (for sadness), 5 (for anger), 13 (for surprise), and 15 (for disgust) loci exhibited main effects on the recognition of facial expressions. Genetic variations in the dopamine system accounted for 3% for fear, 6% for sadness, 7% for anger, 10% for surprise, and 18% for disgust, with the latter surviving a stringent permutation test. Genetic variations in the dopamine system (especially the dopamine synthesis and modulation subsystems) made significant contributions to individual differences in the recognition of disgust faces. Copyright © 2012 S. Karger AG, Basel.

The establishment of Saccharomyces boulardii surface display system using a single expression vector.

PubMed

Wang, Tiantian; Sun, Hui; Zhang, Jie; Liu, Qing; Wang, Longjiang; Chen, Peipei; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin

2014-03-01

In the present study, an a-agglutinin-based Saccharomyces boulardii surface display system was successfully established using a single expression vector. Based on the two protein co-expression vector pSP-G1 built by Partow et al., a S. boulardii surface display vector-pSDSb containing all the display elements was constructed. The display results of heterologous proteins were confirmed by successfully displaying enhanced green fluorescent protein (EGFP) and chicken Eimeria tenella Microneme-2 proteins (EtMic2) on the S. boulardii cell surface. The DNA sequence of AGA1 gene from S. boulardii (SbAGA1) was determined and used as the cell wall anchor partner. This is the first time heterologous proteins have been displayed on the cell surface of S. boulardii. Because S. boulardii is probiotic and eukaryotic, its surface display system would be very valuable, particularly in the development of a live vaccine against various pathogenic organisms especially eukaryotic pathogens such as protistan parasites. Copyright © 2013 Elsevier Inc. All rights reserved.

Efficient biosynthesis of L-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system.

PubMed

Liu, Qiaoli; Zhou, Junping; Yang, Taowei; Zhang, Xian; Xu, Meijuan; Rao, Zhiming

2018-03-01

Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of L-phenylglycine (L-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD + -dependent mutant formate dehydrogenase from Candida boidinii (CbFDH A10C ) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDH A10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was 28.4 mg L -1  min -1  g -1 dry cell weight for L-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L -1 benzoylformic acid was completely converted to pure chiral L-Phg in 4.5 h with 10 g L -1 dry cells and 50.4 g L -1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved L-Phg productivity and demonstrated a novel low-cost method for enantiopure L-Phg production.

Quantitative analysis of Hedgehog gradient formation using an inducible expression system

PubMed Central

Su, Vivian F; Jones, Kelly A; Brodsky, Michael; The, Inge

2007-01-01

Background The Hedgehog (Hh) family of secreted growth factors are morphogens that act in development to direct growth and patterning. Mutations in human Hh and other Hh pathway components have been linked to human diseases. Analysis of Hh distribution during development indicates that cholesterol modification and receptor mediated endocytosis affect the range of Hh signaling and the cellular localization of Hh. Results We have used an inducible, cell type-specific expression system to characterize the three-dimensional distribution of newly synthesized, GFP-tagged Hh in the developing Drosophila wing. Following induction of Hh-GFP expression in posterior producing cells, punctate structures containing Hh-GFP were observed in the anterior target cells. The distance of these particles from the expressing cells was quantified to determine the shape of the Hh gradient at different time points following induction. The majority of cholesterol-modified Hh-GFP was found associated with cells near the anterior/posterior (A/P) boundary, which express high levels of Hh target genes. Without cholesterol, the Hh gradient was flatter, with a lower percentage of particles near the source and a greater maximum distance. Inhibition of Dynamin-dependent endocytosis blocked formation of intracellular Hh particles, but did not prevent movement of newly synthesized Hh to the apical or basolateral surfaces of target cells. In the absence of both cholesterol and endocytosis, Hh particles accumulated in the extracellular space. Staining for the Hh receptor Ptc revealed four categories of Hh particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Interestingly, mainly cholesterol-modified Hh is detected in the cytoplasmic particles lacking Ptc. Conclusion We have developed a system to quantitatively analyze Hh distribution during gradient formation. We directly demonstrate that inhibition of Dynamin-dependent endocytosis is not required for movement of Hh

Quantitative analysis of Hedgehog gradient formation using an inducible expression system.

PubMed

Su, Vivian F; Jones, Kelly A; Brodsky, Michael; The, Inge

2007-05-07

The Hedgehog (Hh) family of secreted growth factors are morphogens that act in development to direct growth and patterning. Mutations in human Hh and other Hh pathway components have been linked to human diseases. Analysis of Hh distribution during development indicates that cholesterol modification and receptor mediated endocytosis affect the range of Hh signaling and the cellular localization of Hh. We have used an inducible, cell type-specific expression system to characterize the three-dimensional distribution of newly synthesized, GFP-tagged Hh in the developing Drosophila wing. Following induction of Hh-GFP expression in posterior producing cells, punctate structures containing Hh-GFP were observed in the anterior target cells. The distance of these particles from the expressing cells was quantified to determine the shape of the Hh gradient at different time points following induction. The majority of cholesterol-modified Hh-GFP was found associated with cells near the anterior/posterior (A/P) boundary, which express high levels of Hh target genes. Without cholesterol, the Hh gradient was flatter, with a lower percentage of particles near the source and a greater maximum distance. Inhibition of Dynamin-dependent endocytosis blocked formation of intracellular Hh particles, but did not prevent movement of newly synthesized Hh to the apical or basolateral surfaces of target cells. In the absence of both cholesterol and endocytosis, Hh particles accumulated in the extracellular space. Staining for the Hh receptor Ptc revealed four categories of Hh particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Interestingly, mainly cholesterol-modified Hh is detected in the cytoplasmic particles lacking Ptc. We have developed a system to quantitatively analyze Hh distribution during gradient formation. We directly demonstrate that inhibition of Dynamin-dependent endocytosis is not required for movement of Hh across target cells, indicating

Role of a Ubiquitously Expressed Receptor in the Vertebrate Olfactory System

PubMed Central

DeMaria, Shannon; Berke, Allison P.; Van Name, Eric; Heravian, Anisa; Ferreira, Todd

2013-01-01

Odorant cues are recognized by receptors expressed on olfactory sensory neurons, the primary sensory neurons of the olfactory epithelium. Odorant receptors typically obey the “one receptor, one neuron” rule, in which the receptive field of the olfactory neuron is determined by the singular odorant receptor that it expresses. Odor-evoked receptor activity across the population of olfactory neurons is then interpreted by the brain to identify the molecular nature of the odorant stimulus. In the present study, we characterized the properties of a C family G-protein-coupled receptor that, unlike most other odorant receptors, is expressed in a large population of microvillous sensory neurons in the zebrafish olfactory epithelium and the mouse vomeronasal organ. We found that this receptor, OlfCc1 in zebrafish and its murine ortholog Vmn2r1, is a calcium-dependent, low-sensitivity receptor specific for the hydrophobic amino acids isoleucine, leucine, and valine. Loss-of-function experiments in zebrafish embryos demonstrate that OlfCc1 is required for olfactory responses to a diverse mixture of polar, nonpolar, acidic, and basic amino acids. OlfCc1 was also found to promote localization of other OlfC receptor family members to the plasma membrane in heterologous cells. Together, these results suggest that the broadly expressed OlfCc1 is required for amino acid detection by the olfactory system and suggest that it plays a role in the function and/or intracellular trafficking of other olfactory and vomeronasal receptors with which it is coexpressed. PMID:24048853

The production of multiprotein complexes in insect cells using the baculovirus expression system.

PubMed

Abdulrahman, Wassim; Radu, Laura; Garzoni, Frederic; Kolesnikova, Olga; Gupta, Kapil; Osz-Papai, Judit; Berger, Imre; Poterszman, Arnaud

2015-01-01

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

Influence of platinum nanoparticles orally administered to rats evaluated by systemic gene expression profiling.

PubMed

Katao, Kazuo; Honma, Reiko; Kato, Satoko; Watanabe, Shinya; Imai, Jun-ichi

2011-01-01

Platinum is recognized as a harmless metal and is widely used in many industrial products. Recent studies have proposed that platinum in the form of nanoparticles has antioxidant properties, suggesting potential uses for platinum nanoparticles as additives in foods and cosmetics, with direct exposure consequences for humans. However, the influence of platinum nanoparticles on humans has not been sufficiently evaluated, thus far. Therefore, to investigate the influence of platinum nanoparticles on a living body, we comprehensively examined the expression profiles of genes obtained from 25 organs and tissues of rats after oral administration of platinum nanoparticles by gavage. Comparative analysis revealed that the expression levels of 18 genes were altered in 12 organs and tissues after the administration (approximately 0.17% of all the genes examined). Of the tissues examined, those of the glandular stomach, which were most directly exposed to the orally administered platinum nanoparticles, showed altered expression levels of genes associated with inflammation. In subcutaneous adipose tissue, the expression levels of genes whose products exhibited ATPase activity were altered. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) analysis confirmed the alteration in the expression levels of these genes in these 2 different tissues. Our findings indicate that orally administered platinum nanoparticles do not have a marked effect on systemic gene expression levels, except on a small number of genes expressed in rat tissues, including peripheral tissues indirectly exposed to the orally administered nanoparticles.

Central nervous system gene expression changes in a transgenic mouse model for bovine spongiform encephalopathy

PubMed Central

2011-01-01

Gene expression analysis has proven to be a very useful tool to gain knowledge of the factors involved in the pathogenesis of diseases, particularly in the initial or preclinical stages. With the aim of finding new data on the events occurring in the Central Nervous System in animals affected with Bovine Spongiform Encephalopathy, a comprehensive genome wide gene expression study was conducted at different time points of the disease on mice genetically modified to model the bovine species brain in terms of cellular prion protein. An accurate analysis of the information generated by microarray technique was the key point to assess the biological relevance of the data obtained in terms of Transmissible Spongiform Encephalopathy pathogenesis. Validation of the microarray technique was achieved by RT-PCR confirming the RNA change and immunohistochemistry techniques that verified that expression changes were translated into variable levels of protein for selected genes. Our study reveals changes in the expression of genes, some of them not previously associated with prion diseases, at early stages of the disease previous to the detection of the pathological prion protein, that might have a role in neuronal degeneration and several transcriptional changes showing an important imbalance in the Central Nervous System homeostasis in advanced stages of the disease. Genes whose expression is altered at early stages of the disease should be considered as possible therapeutic targets and potential disease markers in preclinical diagnostic tool development. Genes non-previously related to prion diseases should be taken into consideration for further investigations. PMID:22035425

Three urocortins in medaka: identification and spatial expression in the central nervous system.

PubMed

Hosono, K; Yamashita, J; Kikuchi, Y; Hiraki-Kajiyama, T; Okubo, K

2017-05-01

The urocortin (UCN) group of neuropeptides includes urocortin 1/sauvagine/urotensin 1 (UTS1), urocortin 2 (UCN2) and urocortin 3 (UCN3). In recent years, evidence has accumulated showing that UCNs play pivotal roles in mediating stress response and anxiety in mammals. Evidence has also emerged regarding the evolutionary conservation of UCNs in vertebrates, but very little information is available about UCNs in non-mammalian vertebrates. Indeed, at present, there are no reports of the empirical identification of ucn2 in non-mammalian vertebrates or of the distribution of ucn2 and ucn3 expression in the adult central nervous system (CNS) of these animals. To gain insight into the evolutionary nature of UCNs in vertebrates, we cloned uts1, ucn2 and ucn3 in a teleost fish, medaka and examined the spatial expression of these genes in the adult brain and spinal cord. Although all known UCN2 genes except those in rodents have been reported to likely lack the necessary structural features to produce a functional pre-pro-protein, all three UCN genes in medaka, including ucn2, displayed all of these features, suggesting their functionality. The three UCN genes exhibited distinct spatial expression patterns in the medaka brain: uts1 was primarily expressed in broad regions of the dorsal telencephalon, ucn2 was expressed in restricted regions of the thalamus and brainstem and ucn3 was expressed in discrete nuclei throughout many regions of the brain. We also found that these genes were all expressed throughout the medaka spinal cord, each with a distinct spatial pattern. Given that many of these regions have been implicated in stress responses and anxiety, the three UCNs may serve distinct physiological roles in the medaka CNS, including those involved in stress and anxiety, as shown in the mammalian CNS. © 2017 British Society for Neuroendocrinology.

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

Interaction between the cholecystokinin and endogenous cannabinoid systems in cued fear expression and extinction retention.

PubMed

Bowers, Mallory E; Ressler, Kerry J

2015-02-01

Post-traumatic stress disorder (PTSD) is thought to develop, in part, from improper inhibition of fear. Accordingly, one of the most effective treatment strategies for PTSD is exposure-based psychotherapy. Ideally, neuroscience would inform adjunct therapies that target the neurotransmitter systems involved in extinction processes. Separate studies have implicated the cholecystokinin (CCK) and endocannabinoid systems in fear; however, there is a high degree of anatomical colocalization between the cannabinoid 1 receptor (Cnr1) and CCK in the basolateral amygdala (BLA), a brain region critical for emotion regulation. Although most research has focused on GABA and GABAergic plasticity as the mechanism by which Cnr1 mediates fear inhibition, we hypothesize that a functional interaction between Cnr1 and CCKB receptor (CCKBR) is critical for fear extinction processes. In this study, systemic pharmacological manipulation of the cannabinoid system modulated cued fear expression in C57BL/6J mice after consolidation of auditory fear conditioning. Knockout of the CCKBR, however, had no effect on fear- or anxiety-like behaviors. Nonetheless, administration of a Cnr1 antagonist increased freezing behavior during a cued fear expression test in wild-type subjects, but had no effect on freezing behavior in CCKBR knockout littermates. In addition, we found that Cnr1-positive fibers form perisomatic clusters around CCKBR-positive cell bodies in the BLA. These CCKBR-positive cells comprise a molecularly heterogenous population of excitatory and inhibitory neurons. These findings provide novel evidence that Cnr1 contributes to cued fear expression via an interaction with the CCK system. Dysfunctional Cnr1-CCKBR interactions might contribute to the etiology of, or result from, fear-related psychiatric disease.

2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori

PubMed Central

Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou

2015-01-01

Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas. PMID:26537835

'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

PubMed

Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

2016-04-01

A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

PubMed Central

Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

2016-01-01

Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system

PubMed Central

Santi, Luca; Giritch, Anatoli; Roy, Chad J.; Marillonnet, Sylvestre; Klimyuk, Victor; Gleba, Yuri; Webb, Robert; Arntzen, Charles J.; Mason, Hugh S.

2006-01-01

Plague is still an endemic disease in different regions of the world. Increasing reports of incidence, the discovery of antibiotic resistance strains, and concern about a potential use of the causative bacteria Yersinia pestis as an agent of biological warfare have highlighted the need for a safe, efficacious, and rapidly producible vaccine. The use of F1 and V antigens and the derived protein fusion F1-V has shown great potential as a protective vaccine in animal studies. Plants have been extensively studied for the production of pharmaceutical proteins as an inexpensive and scalable alternative to common expression systems. In the current study the recombinant plague antigens F1, V, and fusion protein F1-V were produced by transient expression in Nicotiana benthamiana by using a deconstructed tobacco mosaic virus-based system that allowed very rapid and extremely high levels of expression. All of the plant-derived purified antigens, administered s.c. to guinea pigs, generated systemic immune responses and provided protection against an aerosol challenge of virulent Y. pestis. PMID:16410352

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

2015-01-01

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of

Multi-level Expression Design Language: Requirement level (MEDL-R) system evaluation

NASA Technical Reports Server (NTRS)

1980-01-01

An evaluation of the Multi-Level Expression Design Language Requirements Level (MEDL-R) system was conducted to determine whether it would be of use in the Goddard Space Flight Center Code 580 software development environment. The evaluation is based upon a study of the MEDL-R concept of requirement languages, the functions performed by MEDL-R, and the MEDL-R language syntax. Recommendations are made for changes to MEDL-R that would make it useful in the Code 580 environment.

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

PubMed Central

Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C.; Moon, Tae Seok

2016-01-01

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. PMID:26837577

PD1 and PDL1 expression in primary central nervous system diffuse large B-cell lymphoma are frequent and expression of PD1 predicts poor survival.

PubMed

Four, Marion; Cacheux, Valère; Tempier, Ariane; Platero, Dolorès; Fabbro, Michel; Marin, Grégory; Leventoux, Nicolas; Rigau, Valérie; Costes-Martineau, Valérie; Szablewski, Vanessa

2017-12-01

Primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) is a rare and aggressive type of diffuse large B-cell lymphoma (DLBCL) whit poorly understood pathogenesis. Finding biomarkers associated with patient survival may be important for understanding its physiopathology and to develop new therapeutic approaches. We investigated 32 PCNS-DLBCL from immunocompetent patients for BCL2, CMYC, LMO2, and P53 expression and for cytogenetic aberrations of BCL2, BCL6, and MYC genes, all known for their prognostic value in systemic DLBCL (s-DLBCL). We analyzed PD1 and PDL1 protein expression in both tumor infiltrating lymphocytes (TILs) and tumor cells. Finally, we searched for correlation between biological data and clinical course. The PCNS-DLBCL expressed BCL2, CMYC, LMO2, and P53 at similar frequency than s-DLBCL but without significant prognostic on survival. None cases harbored aberrations involving BCL2 and MYC gene whereas BCL6 abnormalities were present in 20.7% of cases but without value on survival. Expression of PD1 in TILs and PDL1 in tumor cells was observed at higher rates than in s-DLBCL (58% and 37%, respectively). The PD1 expression in TILs correlated with PDL1 expression in tumor cells (P = .001). Presence of PD1 positive TILs was associated with poorer overall survival (P = .011). Patients with PDL1 overexpression tended to better response to chemotherapy (P = .23). In conclusion PCNS-DLBCL pathogenesis differs from s-DLBCL without prognostic value of the phenotypic and cytogenetic parameters known for their pejorative impact in the latter. The PD1/PDL1 pathway plays a strong role in PCNS-DLBCL and represents an attractive target for this aggressive lymphoma. Copyright © 2016 John Wiley & Sons, Ltd.

Genetic and epigenetic control of gene expression by CRISPR–Cas systems

PubMed Central

Lo, Albert; Qi, Lei

2017-01-01

The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome. PMID:28649363

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues

PubMed Central

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M.; Zhou, Xinchun

2014-01-01

Aims Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. Methods We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data were then compared in benign and malignant tissues from the same organ/tissue using the student t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. Results We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (either in nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas

Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis

PubMed Central

Scott, RE; Ghule, PN; Stein, JL; Stein, GS

2015-01-01

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e−13 to e−36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. PMID:25808367

Gene Expression Profiling of Peripheral Blood From Kidney Transplant Recipients for the Early Detection of Digestive System Cancer.

PubMed

Kusaka, M; Okamoto, M; Takenaka, M; Sasaki, H; Fukami, N; Kataoka, K; Ito, T; Kenmochi, T; Hoshinaga, K; Shiroki, R

2017-06-01

Kidney transplant recipients are at increased risk of developing cancer in comparison with the general population. To effectively manage post-transplantation malignancies, it is essential to proactively monitor patients. A long-term intensive screening program was associated with a reduced incidence of cancer after transplantation. This study evaluated the usefulness of the gene expression profiling of peripheral blood samples obtained from kidney transplant patients and adopted a screening test for detecting cancer of the digestive system (gastric, colon, pancreas, and biliary tract). Nineteen patients were included in this study and a total of 53 gene expression screening tests were performed. The gene expression profiles of blood-delivered total RNA and whole genome human gene expression profiles were obtained. We investigated the expression levels of 2665 genes associated with digestive cancers and counted the number of genes in which expression was altered. A hierarchical clustering analysis was also performed. The final prediction of the cancer possibility was determined according to an algorithm. The number of genes in which expression was altered was significantly increased in the kidney transplant recipients in comparison with the general population (1091 ± 63 vs 823 ± 94; P = .0024). The number of genes with altered expression decreased after the induction of mechanistic target of rapamycin (mTOR) inhibitor (1484 ± 227 vs 883 ± 154; P = .0439). No cases of possible digestive cancer were detected in this study period. The gene expression profiling of peripheral blood samples may be a useful and noninvasive diagnostic tool that allows for the early detection of cancer of the digestive system. Copyright © 2017 Elsevier Inc. All rights reserved.

Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

PubMed

Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

2009-01-01

Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

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.

Sensitivities of Two Zebrafish TRPA1 Paralogs to Chemical and Thermal Stimuli Analyzed in Heterologous Expression Systems.

PubMed

Oda, Mai; Kurogi, Mako; Kubo, Yoshihiro; Saitoh, Osamu

2016-03-01

Transient receptor potential A1 (TRPA1) is the only member of the mouse, chick, and frog TRPA family, whereas 2 paralogs (zTRPA1a and zTRPA1b) are present in zebrafish. We herein investigated functional differences in the 2 zebrafish TRPA1s. HEK293T cells were used as heterologous expression systems, and the sensitivities of these cells to 4 chemical irritants (allyl isothiocyanate [AITC], caffeine, auto-oxidized epigallocatechin gallate [EGCG], and hydrogen peroxide [H2O2]) were compared with Ca(2+) imaging techniques. Sensitivities to the activators for AITC, oxidized EGCG, and H2O2 were higher in cells expressing zTRPA1a than in those expressing zTRPA1b, whereas caffeine appeared to activate both cells equally. We also characterized the thermal sensitivity of Xenopus oocytes expressing each TRPA1 electrophysiologically using a 2-electrode voltage clamp. Although endogenous currents induced by a cold stimulation were observed in control oocytes in some batches, oocytes expressing zTRPA1b showed significantly stronger cold- and heat-induced responses. However, significant thermal activation was not observed in oocytes expressing zTRPA1a. The results obtained using in vitro expression systems suggest that zTRPA1a is specialized for chemical sensing, whereas zTRPA1b responds to thermal stimuli. Furthermore, characterization of the chimeric molecule of TRPA1a and 1b revealed the importance of the N-terminal region in chemical and thermal sensing by zTRPA1s. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica

DOE PAGES

Shrestha, Roshan P.; Hildebrand, Mark

2017-08-17

An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. In this study, we evaluate a number ofmore » promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.« less

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica

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

Shrestha, Roshan P.; Hildebrand, Mark

An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. In this study, we evaluate a number ofmore » promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.« less

Cancer-specific transgene expression mediated by systemic injection of nanoparticles.

PubMed

Chisholm, Edward J; Vassaux, Georges; Martin-Duque, Pilar; Chevre, Raphael; Lambert, Olivier; Pitard, Bruno; Merron, Andrew; Weeks, Mark; Burnet, Jerome; Peerlinck, Inge; Dai, Ming-Shen; Alusi, Ghassan; Mather, Stephen J; Bolton, Katherine; Uchegbu, Ijeoma F; Schatzlein, Andreas G; Baril, Patrick

2009-03-15

The lack of safe and efficient systemic gene delivery vectors has largely reduced the potential of gene therapy in the clinic. Previously, we have reported that polypropylenimine dendrimer PPIG3/DNA nanoparticles are capable of tumor transfection upon systemic administration in tumor-bearing mice. To be safely applicable in the clinic, it is crucial to investigate the colloidal stability of nanoparticles and to monitor the exact biodistribution of gene transfer in the whole body of the live subject. Our biophysical characterization shows that dendrimers, when complexed with DNA, are capable of forming spontaneously in solution a supramolecular assembly that possesses all the features required to diffuse in experimental tumors through the enhanced permeability and retention effect. We show that these nanoparticles are of sizes ranging from 33 to 286 nm depending on the DNA concentration, with a colloidal stable and well-organized fingerprint-like structure in which DNA molecules are condensed with an even periodicity of 2.8 nm. Whole-body nuclear imaging using small-animal nano-single-photon emission computed tomography/computer tomography scanner and the human Na/I symporter (NIS) as reporter gene shows unique and highly specific tumor targeting with no detection of gene transfer in any of the other tissues of tumor-bearing mice. Tumor-selective transgene expression was confirmed by quantitative reverse transcription-PCR at autopsy of scanned animals, whereas genomic PCR showed that the tumor sites are the predominant sites of nanoparticle accumulation. Considering that NIS imaging of transgene expression has been recently validated in humans, our data highlight the potential of these nanoparticles as a new formulation for cancer gene therapy.

Prolonged Sox4 expression in oligodendrocytes interferes with normal myelination in the central nervous system.

PubMed

Potzner, Michaela R; Griffel, Carola; Lütjen-Drecoll, Elke; Bösl, Michael R; Wegner, Michael; Sock, Elisabeth

2007-08-01

The highly related transcription factors Sox4 and Sox11 are both expressed in oligodendrocyte precursors. Yet whether they have a function in oligodendrocyte development is unknown. By overexpressing Sox4 under the control of 3.1 kb of 5' flanking sequences of the myelin basic protein gene in transgenic mice, we extended Sox4 expression in the oligodendrocyte lineage from oligodendrocyte precursors to cells undergoing terminal differentiation. As a consequence of transgene expression, mice develop the full spectrum of phenotypic traits associated with a severe hypomyelination during the first postnatal weeks. Myelin gene expression was severely reduced, and myelin dramatically thinned in several central nervous system (CNS) regions. Despite these disturbances in CNS myelination, the number of oligodendrocytic cells remained unaltered. Considering that apoptosis rates were normal and proliferation only slightly increased, oligodendrocytes likely persist in a premyelinating to early myelinating state. This shows that prolonged Sox4 expression in cells of the oligodendrocyte lineage is incompatible with the acquisition of a fully mature phenotype and argues that the presence of Sox4, and possibly Sox11, in oligodendrocyte precursors may normally prevent premature differentiation.

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.

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

PubMed

Dettwiler, Ramona; Schmitz, Andrea L; Plattet, Philippe; Zielinski, Jana; Mevissen, Meike

2014-01-01

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94

High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

PubMed

Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

2016-03-01

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

Development of a tightly regulated and highly responsive copper-inducible gene expression system and its application to control of flowering time.

PubMed

Saijo, Takanori; Nagasawa, Akitsu

2014-01-01

A newly developed copper-inducible gene expression system overcame the mixed results reported earlier, worked well both in cultured cells and a whole plant, and enabled to control flowering timing. Copper is one of the essential microelements and is readily taken up by plants. However, to date, it has rarely been used to control the expression of genes of interest, probably due to the inefficiency of the gene expression systems. In this study, we successfully developed a copper-inducible gene expression system that is based on the regulation of the yeast metallothionein gene. This system can be applied in the field and regulated at approximately one-hundredth of the rate used for registered copper-based fungicides. In the presence of copper, a translational fusion of the ACE1 transcription factor with the VP16 activation domain (VP16AD) of herpes simplex virus strongly activated transcription of the GFP gene in transgenic Arabidopsis. Interestingly, insertion of the To71 sequence, a 5'-untranslated region of the 130k/180k gene of tomato mosaic virus, upstream of the GFP gene reduced the basal expression of GFP in the absence of copper to almost negligible levels, even in soil-grown plants that were supplemented with ordinary liquid nutrients. Exposure of plants to 100 μM copper resulted in an over 1,000-fold induction ratio at the transcriptional level of GFP. This induction was copper-specific and dose-dependent with rapid and reversible responses. Using this expression system, we also succeeded in regulating floral transition by copper treatment. These results indicate that our newly developed copper-inducible system can accelerate gene functional analysis in model plants and can be used to generate novel agronomic traits in crop species.

Close-form expression of one-tap normalized LMS carrier phase recovery in optical communication systems

NASA Astrophysics Data System (ADS)

Xu, Tianhua; Jacobsen, Gunnar; Popov, Sergei; Li, Jie; Liu, Tiegen; Zhang, Yimo

2016-10-01

The performance of long-haul high speed coherent optical fiber communication systems is significantly degraded by the laser phase noise and the equalization enhanced phase noise (EEPN). In this paper, the analysis of the one-tap normalized least-mean-square (LMS) carrier phase recovery (CPR) is carried out and the close-form expression is investigated for quadrature phase shift keying (QPSK) coherent optical fiber communication systems, in compensating both laser phase noise and equalization enhanced phase noise. Numerical simulations have also been implemented to verify the theoretical analysis. It is found that the one-tap normalized least-mean-square algorithm gives the same analytical expression for predicting CPR bit-error-rate (BER) floors as the traditional differential carrier phase recovery, when both the laser phase noise and the equalization enhanced phase noise are taken into account.

Expression, localization and possible functions of aquaporins 3 and 8 in rat digestive system.

PubMed

Zhao, G X; Dong, P P; Peng, R; Li, J; Zhang, D Y; Wang, J Y; Shen, X Z; Dong, L; Sun, J Y

2016-01-01

Although aquaporins (AQPs) play important roles in transcellular water movement, their precise quantification and localization remains controversial. We investigated expression levels and localizations of AQP3 and AQP8 and their possible functions in the rat digestive system using real-time polymerase chain reactions, western blot analysis and immunohistochemistry. We investigated the expression levels and localizations of AQP3 and AQP8 in esophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, proximal and distal colon, and liver. AQP3 was expressed in the basolateral membranes of stratified epithelia (esophagus and forestomach) and simple columnar epithelia (glandular stomach, ileum, and proximal and distal colon). Expression was particularly abundant in the esophagus, and proximal and distal colon. AQP8 was found in the subapical compartment of columnar epithelial cells of the jejunum, ileum, proximal colon and liver; the most intense staining occurred in the jejunum. Our results suggest that AQP3 and AQP8 play significant roles in intestinal function and/or fluid homeostasis and may be an important subject for future investigation of disorders that involve disruption of intestinal fluid homeostasis, such as inflammatory bowel disease and irritable bowel syndrome.

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.

A computational model of the development of separate representations of facial identity and expression in the primate visual system.

PubMed

Tromans, James Matthew; Harris, Mitchell; Stringer, Simon Maitland

2011-01-01

Experimental studies have provided evidence that the visual processing areas of the primate brain represent facial identity and facial expression within different subpopulations of neurons. For example, in non-human primates there is evidence that cells within the inferior temporal gyrus (TE) respond primarily to facial identity, while cells within the superior temporal sulcus (STS) respond to facial expression. More recently, it has been found that the orbitofrontal cortex (OFC) of non-human primates contains some cells that respond exclusively to changes in facial identity, while other cells respond exclusively to facial expression. How might the primate visual system develop physically separate representations of facial identity and expression given that the visual system is always exposed to simultaneous combinations of facial identity and expression during learning? In this paper, a biologically plausible neural network model, VisNet, of the ventral visual pathway is trained on a set of carefully-designed cartoon faces with different identities and expressions. The VisNet model architecture is composed of a hierarchical series of four Self-Organising Maps (SOMs), with associative learning in the feedforward synaptic connections between successive layers. During learning, the network develops separate clusters of cells that respond exclusively to either facial identity or facial expression. We interpret the performance of the network in terms of the learning properties of SOMs, which are able to exploit the statistical indendependence between facial identity and expression.

Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

PubMed Central

2012-01-01

Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared

Vancomycin modifies the expression of the agr system in multidrug-resistant Staphylococcus aureus clinical isolates

PubMed Central

Cázares-Domínguez, Vicenta; Ochoa, Sara A.; Cruz-Córdova, Ariadnna; Rodea, Gerardo E.; Escalona, Gerardo; Olivares, Alma L.; Olivares-Trejo, José de Jesús; Velázquez-Guadarrama, Norma; Xicohtencatl-Cortes, Juan

2015-01-01

Staphylococcus aureus is an opportunistic pathogen that colonizes human hosts and causes a wide variety of diseases. Two interacting regulatory systems called agr (accessory gene regulator) and sar (staphylococcal accessory regulator) are involved in the regulation of virulence factors. The aim of this study was to evaluate the effect of vancomycin on hld and spa gene expression during the exponential and post-exponential growth phases in multidrug-resistant (MDR) S. aureus. Methods: Antibiotic susceptibility was evaluated by the standard microdilution method. The phylogenetic profile was obtained by pulsed-field gel electrophoresis (PFGE). Polymorphisms of agr and SCCmec (staphylococcal cassette chromosome mec) were analyzed by multiplex polymerase chain reaction (PCR). The expression levels of hld and spa were analyzed by reverse transcription-PCR. An enzyme-linked immunosorbent assay (ELISA) was performed to detect protein A, and biofilm formation was analyzed via crystal violet staining. Results: In total, 60.60% (20/33) of S. aureus clinical isolates were MDR. Half (10/20) of the MDR S. aureus isolates were distributed in subcluster 10, with >90% similarity among them. In the isolates of this subcluster, a high prevalence (100%) for the agrII and the cassette SCCmec II polymorphisms was found. Our data showed significant increases in hld expression during the post-exponential phase in the presence and absence of vancomycin. Significant increases in spa expression, protein A production and biofilm formation were observed during the post-exponential phase when the MDR S. aureus isolates were challenged with vancomycin. Conclusion: The polymorphism agrII, which is associated with nosocomial isolates, was the most prevalent polymorphism in MDR S. aureus. Additionally, under our study conditions, vancomycin modified hld and spa expression in these clinical isolates. Therefore, vancomycin may regulate alternative systems that jointly participate in the regulation of

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

PubMed

Buyel, Johannes Felix; Fischer, Rainer

2014-01-31

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

Dual Luciferase Assay System for Rapid Assessment of Gene Expression in Saccharomyces cerevisiae

PubMed Central

McNabb, David S.; Reed, Robin; Marciniak, Robert A.

2005-01-01

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements. PMID:16151247

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

PubMed

Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

2016-03-18

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

PubMed

Wang, Chunyi; Mao, Jinghe; Redfield, Samantha; Mo, Yinyuan; Lage, Janice M; Zhou, Xinchun

2014-10-01

Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft

Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.

PubMed

Davis, J Q; Bennett, V

1993-01-01

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

CD30 expression by bone marrow mast cells from different diagnostic variants of systemic mastocytosis.

PubMed

Morgado, José M; Perbellini, Omar; Johnson, Ryan C; Teodósio, Cristina; Matito, Almudena; Álvarez-Twose, Iván; Bonadonna, Patrizia; Zamò, Alberto; Jara-Acevedo, Maria; Mayado, Andrea; Garcia-Montero, Andrés; Mollejo, Manuela; George, Tracy I; Zanotti, Roberta; Orfao, Alberto; Escribano, Luis; Sánchez-Muñoz, Laura

2013-12-01

CD30 expression by bone marrow (BM) mast cells (MC) has been reported recently in systemic mastocytosis (SM) patients. The aim of this study was to investigate the potential diagnostic and prognostic value of CD30 expression in SM as assessed by multiparameter flow cytometry. A total of 163 consecutive BM samples corresponding to 142 SM patients and 21 non-mastocytosis cases were studied. CD30 was positive in most SM patients (80%), but in only one non-mastocytosis case (4.8%). When combined with CD25, CD30 contributed to an improved accuracy over that of CD25 alone (98% versus 93%) mainly because most (eight of nine) of the well-differentiated SM (WDSM), who lacked CD25, were CD30(+). Similar levels of expression of CD30 were observed among all different subgroups of SM except mast cell leukaemia; among indolent SM (ISM) patients, no significant association was observed between the levels of CD30 expression and other clinical and biological features of the disease. The increased expression of CD30 associated with absence of CD25 contributes to the diagnosis of WDSM and its distinction from other subtypes of SM. By contrast, CD30 expression did not contribute either to prognostic stratification of ISM or to the differential diagnosis between ISM and aggressive SM cases. © 2013 John Wiley & Sons Ltd.

Recognizing Action Units for Facial Expression Analysis

PubMed Central

Tian, Ying-li; Kanade, Takeo; Cohn, Jeffrey F.

2010-01-01

Most automatic expression analysis systems attempt to recognize a small set of prototypic expressions, such as happiness, anger, surprise, and fear. Such prototypic expressions, however, occur rather infrequently. Human emotions and intentions are more often communicated by changes in one or a few discrete facial features. In this paper, we develop an Automatic Face Analysis (AFA) system to analyze facial expressions based on both permanent facial features (brows, eyes, mouth) and transient facial features (deepening of facial furrows) in a nearly frontal-view face image sequence. The AFA system recognizes fine-grained changes in facial expression into action units (AUs) of the Facial Action Coding System (FACS), instead of a few prototypic expressions. Multistate face and facial component models are proposed for tracking and modeling the various facial features, including lips, eyes, brows, cheeks, and furrows. During tracking, detailed parametric descriptions of the facial features are extracted. With these parameters as the inputs, a group of action units (neutral expression, six upper face AUs and 10 lower face AUs) are recognized whether they occur alone or in combinations. The system has achieved average recognition rates of 96.4 percent (95.4 percent if neutral expressions are excluded) for upper face AUs and 96.7 percent (95.6 percent with neutral expressions excluded) for lower face AUs. The generalizability of the system has been tested by using independent image databases collected and FACS-coded for ground-truth by different research teams. PMID:25210210

A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

PubMed Central

Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David

2013-01-01

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852

Association of large intergenic noncoding RNA expression with disease activity and organ damage in systemic lupus erythematosus.

PubMed

Wu, Yanfang; Zhang, Feifei; Ma, Jianyang; Zhang, Xiaoyan; Wu, Lingling; Qu, Bo; Xia, Shiwei; Chen, Shunle; Tang, Yuanjia; Shen, Nan

2015-05-21

Despite growing evidence that large intergenic noncoding RNAs (lincRNAs) can regulate gene expression and widely take part in normal physiological and disease conditions, our knowledge of systemic lupus erythematosus (SLE)-related lincRNAs remains limited. The aim of this study was to detect the levels of four lincRNAs (ENST00000500949: linc0949, ENST00000500597: linc0597, ENST00000501992: linc1992, and ENST00000523995: linc3995) involved in innate immunity in the peripheral blood mononuclear cells (PBMCs) of patients with SLE and correlate these lincRNA levels with disease activity, organ damage, clinical features and medical therapies. PBMCs were obtained from 102 patients with SLE, 54 patients with rheumatoid arthritis (RA) and 76 healthy donors. lincRNA expression levels were measured by real-time quantitative polymerase chain reaction. Disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) scores, and organ damage was evaluated with the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. linc0949 and linc0597 were significantly decreased in patients with SLE compared with patients with RA and healthy control subjects. linc0949 was correlated with SLEDAI-2K score (r = -0.329, P = 0.0007), as well as with complement component C3 level (r = 0.348, P = 0.0003). The level of linc0949 was also reduced in patients with SLE who had the presence of cumulative organ damage. In addition, decreasing expression of linc0949 was associated with lupus nephritis. linc0949 expression significantly increased after treatment, whereas neither disease activity nor organ damage correlated with linc0597 expression. Our results provide novel empirical evidence that linc0949 could be a potential biomarker for diagnosis, disease activity and therapeutic response in SLE.

Bioinformatics analysis of differentially expressed gene profiles associated with systemic lupus erythematosus

PubMed Central

Wu, Chengjiang; Zhao, Yangjing; Lin, Yu; Yang, Xinxin; Yan, Meina; Min, Yujiao; Pan, Zihui; Xia, Sheng; Shao, Qixiang

2018-01-01

DNA microarray and high-throughput sequencing have been widely used to identify the differentially expressed genes (DEGs) in systemic lupus erythematosus (SLE). However, the big data from gene microarrays are also challenging to work with in terms of analysis and processing. The presents study combined data from the microarray expression profile (GSE65391) and bioinformatics analysis to identify the key genes and cellular pathways in SLE. Gene ontology (GO) and cellular pathway enrichment analyses of DEGs were performed to investigate significantly enriched pathways. A protein-protein interaction network was constructed to determine the key genes in the occurrence and development of SLE. A total of 310 DEGs were identified in SLE, including 193 upregulated genes and 117 downregulated genes. GO analysis revealed that the most significant biological process of DEGs was immune system process. Kyoto Encyclopedia of Genes and Genome pathway analysis showed that these DEGs were enriched in signaling pathways associated with the immune system, including the RIG-I-like receptor signaling pathway, intestinal immune network for IgA production, antigen processing and presentation and the toll-like receptor signaling pathway. The current study screened the top 10 genes with higher degrees as hub genes, which included 2′-5′-oligoadenylate synthetase 1, MX dynamin like GTPase 2, interferon induced protein with tetratricopeptide repeats 1, interferon regulatory factor 7, interferon induced with helicase C domain 1, signal transducer and activator of transcription 1, ISG15 ubiquitin-like modifier, DExD/H-box helicase 58, interferon induced protein with tetratricopeptide repeats 3 and 2′-5′-oligoadenylate synthetase 2. Module analysis revealed that these hub genes were also involved in the RIG-I-like receptor signaling, cytosolic DNA-sensing, toll-like receptor signaling and ribosome biogenesis pathways. In addition, these hub genes, from different probe sets, exhibited

A putative vesicular transporter expressed in Drosophila mushroom bodies that mediates sexual behavior may define a novel neurotransmitter system

PubMed Central

Brooks, Elizabeth S.; Greer, Christina L.; Romero-Calderón, Rafael; Serway, Christine N.; Grygoruk, Anna; Haimovitz, Jasmine M.; Nguyen, Bac T.; Najibi, Rod; Tabone, Christopher J.; de Belle, J. Steven; Krantz, David E.

2011-01-01

Summary Storage and release of classical and amino acid neurotransmitters requires vesicular transporters. Some neurons lack known vesicular transporters, suggesting additional neurotransmitter systems remain unidentified. Insect mushroom bodies (MBs) are critical for several behaviors, including learning, but the neurotransmitters released by the intrinsic Kenyon cells (KCs) remain unknown. Likewise, KCs do not express a known vesicular transporter. We report the identification of a novel Drosophila gene portabella (prt) that is structurally similar to known vesicular transporters. Both larval and adult brains express PRT in the KCs of the MBs. Additional PRT cells project to the central complex and optic ganglia. prt mutation causes an olfactory learning deficit and an unusual defect in the male’s position during copulation that is rescued by expression in KCs. Since prt is expressed in neurons that lack other known vesicular transporters or neurotransmitters, it may define a previously unknown neurotransmitter system responsible for sexual behavior and a component of olfactory learning. PMID:22017990

Dynamic gene expression in the song system of zebra finches during the song learning period.

PubMed

Olson, Christopher R; Hodges, Lisa K; Mello, Claudio V

2015-12-01

The brain circuitry that controls song learning and production undergoes marked changes in morphology and connectivity during the song learning period in juvenile zebra finches, in parallel to the acquisition, practice and refinement of song. Yet, the genetic programs and timing of regulatory change that establish the neuronal connectivity and plasticity during this critical learning period remain largely undetermined. To address this question, we used in situ hybridization to compare the expression patterns of a set of 30 known robust molecular markers of HVC and/or area X, major telencephalic song nuclei, between adult and juvenile male zebra finches at different ages during development (20, 35, 50 days post-hatch, dph). We found that several of the genes examined undergo substantial changes in expression within HVC or its surrounds, and/or in other song nuclei. They fit into broad patterns of regulation, including those whose expression within HVC during this period increases (COL12A1, COL 21A1, MPZL1, PVALB, and CXCR7) or decreases (e.g., KCNT2, SAP30L), as well as some that show decreased expression in the surrounding tissue with little change within song nuclei (e.g. SV2B, TAC1). These results reveal a broad range of molecular changes that occur in the song system in concert with the song learning period. Some of the genes and pathways identified are potential modulators of the developmental changes associated with the emergence of the adult properties of the song control system, and/or the acquisition of learned vocalizations in songbirds. © 2015 Wiley Periodicals, Inc.

MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system

PubMed Central

Kapsimali, Marika; Kloosterman, Wigard P; de Bruijn, Ewart; Rosa, Frederic; Plasterk, Ronald HA; Wilson, Stephen W

2007-01-01

Background MicroRNA (miRNA) encoding genes are abundant in vertebrate genomes but very few have been studied in any detail. Bioinformatic tools allow prediction of miRNA targets and this information coupled with knowledge of miRNA expression profiles facilitates formulation of hypotheses of miRNA function. Although the central nervous system (CNS) is a prominent site of miRNA expression, virtually nothing is known about the spatial and temporal expression profiles of miRNAs in the brain. To provide an overview of the breadth of miRNA expression in the CNS, we performed a comprehensive analysis of the neuroanatomical expression profiles of 38 abundant conserved miRNAs in developing and adult zebrafish brain. Results Our results show miRNAs have a wide variety of different expression profiles in neural cells, including: expression in neuronal precursors and stem cells (for example, miR-92b); expression associated with transition from proliferation to differentiation (for example, miR-124); constitutive expression in mature neurons (miR-124 again); expression in both proliferative cells and their differentiated progeny (for example, miR-9); regionally restricted expression (for example, miR-222 in telencephalon); and cell-type specific expression (for example, miR-218a in motor neurons). Conclusion The data we present facilitate prediction of likely modes of miRNA function in the CNS and many miRNA expression profiles are consistent with the mutual exclusion mode of function in which there is spatial or temporal exclusion of miRNAs and their targets. However, some miRNAs, such as those with cell-type specific expression, are more likely to be co-expressed with their targets. Our data provide an important resource for future functional studies of miRNAs in the CNS. PMID:17711588

Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

PubMed

Takala, T M; Saris, P E J; Tynkkynen, S S H

2003-01-01

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

Quantification of protein expression in cells and cellular subcompartments on immunohistochemical sections using a computer supported image analysis system.

PubMed

Braun, Martin; Kirsten, Robert; Rupp, Niels J; Moch, Holger; Fend, Falko; Wernert, Nicolas; Kristiansen, Glen; Perner, Sven

2013-05-01

Quantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. Several manual ('eyeballing') scoring systems are used in order to semi-quantify protein expression based on chromogenic intensities and distribution patterns. However, manual scoring systems are time-consuming and subject to significant intra- and interobserver variability. The aim of our study was to explore, whether new image analysis software proves to be sufficient as an alternative tool to quantify protein expression. For IHC experiments, one nucleus specific marker (i.e., ERG antibody), one cytoplasmic specific marker (i.e., SLC45A3 antibody), and one marker expressed in both compartments (i.e., TMPRSS2 antibody) were chosen. Stainings were applied on TMAs, containing tumor material of 630 prostate cancer patients. A pathologist visually quantified all IHC stainings in a blinded manner, applying a four-step scoring system. For digital quantification, image analysis software (Tissue Studio v.2.1, Definiens AG, Munich, Germany) was applied to obtain a continuous spectrum of average staining intensity. For each of the three antibodies we found a strong correlation of the manual protein expression score and the score of the image analysis software. Spearman's rank correlation coefficient was 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively (p⟨0.01). Our data suggest that the image analysis software Tissue Studio is a powerful tool for quantification of protein expression in IHC stainings. Further, since the digital analysis is precise and reproducible, computer supported protein quantification might help to overcome intra- and interobserver variability and increase objectivity of IHC based protein assessment.

Reconstructing dynamic mental models of facial expressions in prosopagnosia reveals distinct representations for identity and expression.

PubMed

Richoz, Anne-Raphaëlle; Jack, Rachael E; Garrod, Oliver G B; Schyns, Philippe G; Caldara, Roberto

2015-04-01

The human face transmits a wealth of signals that readily provide crucial information for social interactions, such as facial identity and emotional expression. Yet, a fundamental question remains unresolved: does the face information for identity and emotional expression categorization tap into common or distinct representational systems? To address this question we tested PS, a pure case of acquired prosopagnosia with bilateral occipitotemporal lesions anatomically sparing the regions that are assumed to contribute to facial expression (de)coding (i.e., the amygdala, the insula and the posterior superior temporal sulcus--pSTS). We previously demonstrated that PS does not use information from the eye region to identify faces, but relies on the suboptimal mouth region. PS's abnormal information use for identity, coupled with her neural dissociation, provides a unique opportunity to probe the existence of a dichotomy in the face representational system. To reconstruct the mental models of the six basic facial expressions of emotion in PS and age-matched healthy observers, we used a novel reverse correlation technique tracking information use on dynamic faces. PS was comparable to controls, using all facial features to (de)code facial expressions with the exception of fear. PS's normal (de)coding of dynamic facial expressions suggests that the face system relies either on distinct representational systems for identity and expression, or dissociable cortical pathways to access them. Interestingly, PS showed a selective impairment for categorizing many static facial expressions, which could be accounted for by her lesion in the right inferior occipital gyrus. PS's advantage for dynamic facial expressions might instead relate to a functionally distinct and sufficient cortical pathway directly connecting the early visual cortex to the spared pSTS. Altogether, our data provide critical insights on the healthy and impaired face systems, question evidence of deficits

A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria.

PubMed

Hennessy, Rosanna C; Christiansen, Line; Olsson, Stefan; Stougaard, Peter

2018-01-01

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. Here we describe a dual-fluorescence reporter system carrying the red fluorescent marker mCherry and the blue fluorescent protein EBFP2 enabling the simultaneous analysis of two promoters in broad-host range autofluorescent Gram-negative bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum

PubMed Central

Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; dos Santos, Jessica Brandão; Pacheco, Fabiana dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

2016-01-01

Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates—a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

Expression of CD64 on Circulating Neutrophils Favoring Systemic Inflammatory Status in Erythema Nodosum Leprosum.

PubMed

Schmitz, Veronica; Prata, Rhana Berto da Silva; Barbosa, Mayara Garcia de Mattos; Mendes, Mayara Abud; Brandão, Sheila Santos; Amadeu, Thaís Porto; Rodrigues, Luciana Silva; Ferreira, Helen; Costa, Fabrício da Mota Ramalho; Dos Santos, Jessica Brandão; Pacheco, Fabiana Dos Santos; Machado, Alice de Miranda; Nery, José Augusto da Costa; Hacker, Mariana de Andrea; Sales, Anna Maria; Pinheiro, Roberta Olmo; Sarno, Euzenir Nunes

2016-08-01

Erythema Nodosum Leprosum (ENL) is an immune reaction in leprosy that aggravates the patient´s clinical condition. ENL presents systemic symptoms of an acute infectious syndrome with high leukocytosis and intense malaise clinically similar to sepsis. The treatment of ENL patients requires immunosuppression and thus needs to be early and efficient to prevent both disabilities and permanent nerve damage. Some patients experience multiple episodes of ENL and prolonged use of immunosuppressive drugs may lead to serious adverse effects. Thalidomide treatment is extremely effective at ameliorating ENL symptoms. Several mechanisms have been proposed to explain the efficacy of thalidomide in ENL, including the inhibition of TNF production. Given its teratogenicity, thalidomide is prohibitive for women of childbearing age. A rational search for molecular targets during ENL episodes is essential to better understand the disease mechanisms involved, which may also lead to the discovery of new drugs and diagnostic tests. Previous studies have demonstrated that IFN-γ and GM-CSF, involved in the induction of CD64 expression, increase during ENL. The aim of the present study was to investigate CD64 expression during ENL and whether thalidomide treatment modulated its expression. Leprosy patients were allocated to one of five groups: (1) Lepromatous leprosy, (2) Borderline leprosy, (3) Reversal reaction, (4) ENL, and (5) ENL 7 days after thalidomide treatment. The present study demonstrated that CD64 mRNA and protein were expressed in ENL lesions and that thalidomide treatment reduced CD64 expression and neutrophil infiltrates-a hallmark of ENL. We also showed that ENL blood neutrophils exclusively expressed CD64 on the cell surface and that thalidomide diminished overall expression. Patient classification based on clinical symptoms found that severe ENL presented high levels of neutrophil CD64. Collectively, these data revealed that ENL neutrophils express CD64, presumably

Facial expressions as a model to test the role of the sensorimotor system in the visual perception of the actions.

PubMed

Mele, Sonia; Ghirardi, Valentina; Craighero, Laila

2017-12-01

A long-term debate concerns whether the sensorimotor coding carried out during transitive actions observation reflects the low-level movement implementation details or the movement goals. On the contrary, phonemes and emotional facial expressions are intransitive actions that do not fall into this debate. The investigation of phonemes discrimination has proven to be a good model to demonstrate that the sensorimotor system plays a role in understanding actions acoustically presented. In the present study, we adapted the experimental paradigms already used in phonemes discrimination during face posture manipulation, to the discrimination of emotional facial expressions. We submitted participants to a lower or to an upper face posture manipulation during the execution of a four alternative labelling task of pictures randomly taken from four morphed continua between two emotional facial expressions. The results showed that the implementation of low-level movement details influence the discrimination of ambiguous facial expressions differing for a specific involvement of those movement details. These findings indicate that facial expressions discrimination is a good model to test the role of the sensorimotor system in the perception of actions visually presented.

Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 11; Express/T-160E Project Express A2 and A3 Data Agreement Document

NASA Technical Reports Server (NTRS)

Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.; Dunning, John (Technical Monitor)

2003-01-01

This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80deg E. and 11deg W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3-99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 12; Express/T-160 Project Express A2 and A3 Sensors Operations Procedures Document

NASA Technical Reports Server (NTRS)

Dunning, John (Technical Monitor); Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

2003-01-01

This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 deg. E. and 11 deg. W respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

Transient Expression of Lumbrokinase (PI239) in Tobacco (Nicotiana tabacum) Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots.

PubMed

Dickey, Alexia; Wang, Nan; Cooper, Edwin; Tull, Lauren; Breedlove, Drew; Mason, Hugh; Liu, Dehu; Wang, Kevin Yueju

2017-01-01

Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

Heterologous Expression of Equine CYP3A94 and Investigation of a Tunable System to Regulate Co-Expressed NADPH P450 Oxidoreductase Levels

PubMed Central

Dettwiler, Ramona; Schmitz, Andrea L.; Plattet, Philippe; Zielinski, Jana; Mevissen, Meike

2014-01-01

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of “Shield-1” prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A

A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei.

PubMed

Lu, W W; Wang, T; Wang, Y; Xin, M; Kong, J

2016-03-01

Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection.

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.

Pose-variant facial expression recognition using an embedded image system

NASA Astrophysics Data System (ADS)

Song, Kai-Tai; Han, Meng-Ju; Chang, Shuo-Hung

2008-12-01

In recent years, one of the most attractive research areas in human-robot interaction is automated facial expression recognition. Through recognizing the facial expression, a pet robot can interact with human in a more natural manner. In this study, we focus on the facial pose-variant problem. A novel method is proposed in this paper to recognize pose-variant facial expressions. After locating the face position in an image frame, the active appearance model (AAM) is applied to track facial features. Fourteen feature points are extracted to represent the variation of facial expressions. The distance between feature points are defined as the feature values. These feature values are sent to a support vector machine (SVM) for facial expression determination. The pose-variant facial expression is classified into happiness, neutral, sadness, surprise or anger. Furthermore, in order to evaluate the performance for practical applications, this study also built a low resolution database (160x120 pixels) using a CMOS image sensor. Experimental results show that the recognition rate is 84% with the self-built database.

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

PubMed

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

2016-05-25

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

Mosaic expression of Atrx in the mouse central nervous system causes memory deficits

PubMed Central

Tamming, Renee J.; Siu, Jennifer R.; Jiang, Yan; Prado, Marco A. M.; Beier, Frank

2017-01-01

ABSTRACT The rapid modulation of chromatin organization is thought to play a crucial role in cognitive processes such as memory consolidation. This is supported in part by the dysregulation of many chromatin-remodelling proteins in neurodevelopmental and psychiatric disorders. A key example is ATRX, an X-linked gene commonly mutated in individuals with syndromic and nonsyndromic intellectual disability. The consequences of Atrx inactivation for learning and memory have been difficult to evaluate because of the early lethality of hemizygous-null animals. In this study, we evaluated the outcome of brain-specific Atrx deletion in heterozygous female mice. These mice exhibit a mosaic pattern of ATRX protein expression in the central nervous system attributable to the location of the gene on the X chromosome. Although the hemizygous male mice die soon after birth, heterozygous females survive to adulthood. Body growth is stunted in these animals, and they have low circulating concentrations of insulin growth factor 1. In addition, they are impaired in spatial, contextual fear and novel object recognition memory. Our findings demonstrate that mosaic loss of ATRX expression in the central nervous system leads to endocrine defects and decreased body size and has a negative impact on learning and memory. PMID:28093507

Mosaic expression of Atrx in the mouse central nervous system causes memory deficits.

PubMed

Tamming, Renee J; Siu, Jennifer R; Jiang, Yan; Prado, Marco A M; Beier, Frank; Bérubé, Nathalie G

2017-02-01

The rapid modulation of chromatin organization is thought to play a crucial role in cognitive processes such as memory consolidation. This is supported in part by the dysregulation of many chromatin-remodelling proteins in neurodevelopmental and psychiatric disorders. A key example is ATRX, an X-linked gene commonly mutated in individuals with syndromic and nonsyndromic intellectual disability. The consequences of Atrx inactivation for learning and memory have been difficult to evaluate because of the early lethality of hemizygous-null animals. In this study, we evaluated the outcome of brain-specific Atrx deletion in heterozygous female mice. These mice exhibit a mosaic pattern of ATRX protein expression in the central nervous system attributable to the location of the gene on the X chromosome. Although the hemizygous male mice die soon after birth, heterozygous females survive to adulthood. Body growth is stunted in these animals, and they have low circulating concentrations of insulin growth factor 1. In addition, they are impaired in spatial, contextual fear and novel object recognition memory. Our findings demonstrate that mosaic loss of ATRX expression in the central nervous system leads to endocrine defects and decreased body size and has a negative impact on learning and memory. © 2017. Published by The Company of Biologists Ltd.

Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct.

PubMed

Yu, Ming-Jiun; Miller, R Lance; Uawithya, Panapat; Rinschen, Markus M; Khositseth, Sookkasem; Braucht, Drew W W; Chou, Chung-Lin; Pisitkun, Trairak; Nelson, Raoul D; Knepper, Mark A

2009-02-17

We used a systems biology-based approach to investigate the basis of cell-specific expression of the water channel aquaporin-2 (AQP2) in the renal collecting duct. Computational analysis of the 5'-flanking region of the AQP2 gene (Genomatix) revealed 2 conserved clusters of putative transcriptional regulator (TR) binding elements (BEs) centered at -513 bp (corresponding to the SF1, NFAT, and FKHD TR families) and -224 bp (corresponding to the AP2, SRF, CREB, GATA, and HOX TR families). Three other conserved motifs corresponded to the ETS, EBOX, and RXR TR families. To identify TRs that potentially bind to these BEs, we carried out mRNA profiling (Affymetrix) in mouse mpkCCDc14 collecting duct cells, revealing expression of 25 TRs that are also expressed in native inner medullary collecting duct. One showed a significant positive correlation with AQP2 mRNA abundance among mpkCCD subclones (Ets1), and 2 showed a significant negative correlation (Elf1 and an orphan nuclear receptor Nr1h2). Transcriptomic profiling in native proximal tubules (PT), medullary thick ascending limbs (MTAL), and IMCDs from kidney identified 14 TRs (including Ets1 and HoxD3) expressed in the IMCD but not PT or MTAL (candidate AQP2 enhancer roles), and 5 TRs (including HoxA5, HoxA9 and HoxA10) expressed in PT and MTAL but not in IMCD (candidate AQP2 repressor roles). In luciferase reporter assays, overexpression of 3 ETS family TRs transactivated the mouse proximal AQP2 promoter. The results implicate ETS family TRs in cell-specific expression of AQP2 and point to HOX, RXR, CREB and GATA family TRs as playing likely additional roles.

A self-initiating eukaryotic transient gene expression system based on contransfection of bacteriophage T7 RNA polymerase and DNA vectors containing a T7 autogene.

PubMed Central

Chen, X; Li, Y; Xiong, K; Wagner, T E

1994-01-01

A novel cytoplasmic gene expression system has been developed. This system differs from other expression systems in that it relies on the co-delivery of plasmid DNA and T7 RNA polymerase (RNAP) during transfection. The plasmid contains a T7 RNAP gene driven by the T7 promoter (T7 autogene) and a functional/reporter gene driven by another T7 promoter (T7T7/T7-gene construct). Once this DNA-enzyme complex is introduced into eukaryotic cells, the transcription of the T7 RNAP and the functional/reporter genes is initiated by the co-delivered T7 RNAP. The T7 RNAP, which is responsible for the initiation and maintenance of expression of both T7 and functional/reporter genes, is replenished by translation of newly synthesized T7 mRNA. This T7 system was designed in such a manner that the expression of the functional/reporter genes can occur in the cytoplasm and does not require any nuclear involvement. When transfected by either a pT7T7/T7Luc or a pT7T7/T7hGH plasmids with the cointroduced T7 RNAP, mouse L cells were found to express high levels of luciferase immediately after transfection, apparently due to the cytoplasmic gene expression; the expression of human growth hormone (hGH) could be sustained for at least 6 days. Both T7 and hGH mRNA were expressed by the cells transfected with pT7T7/T7hGH. These results suggest that this cytoplasmic expression system may be used for certain targets of somatic gene therapy. Images PMID:8029020

Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species.

PubMed

Liu, Yanbin; Koh, Chong Mei John; Ngoh, Si Te; Ji, Lianghui

2015-10-26

Rhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts. We have developed a luciferase gene reporter assay based on a new codon-optimized LUC2 reporter gene (RtLUC2), which is flanked with CAR2 homology arms and can be integrated into the CAR2 locus in the nuclear genome at >90 % efficiency. We characterized the upstream DNA sequence of a D-amino acid oxidase gene (DAO1) from R. toruloides ATCC 10657 by nested deletions. By comparing the upstream DNA sequences of several putative DAO1 homologs of Basidiomycetous fungi, we identified a conserved DNA motif with a consensus sequence of AGGXXGXAGX11GAXGAXGG within a 0.2 kb region from the mRNA translation initiation site. Deletion of this motif led to strong mRNA transcription under non-inducing conditions. Interestingly, DAO1 promoter activity was enhanced about fivefold when the 108 bp intron 1 was included in the reporter construct. We identified a conserved CT-rich motif in the intron with a consensus sequence of TYTCCCYCTCCYCCCCACWYCCGA, deletion or point mutations of which drastically reduced promoter strength under both inducing and non-inducing conditions. Additionally, we created a selection marker-free DAO1-null mutant (∆dao1e) which displayed greatly improved inducible gene expression, particularly when both glucose and nitrogen were present in high levels. To avoid adding unwanted peptide to proteins to be expressed, we converted the original translation initiation codon to ATC and re-created a translation initiation codon at the start of exon 2. This promoter, named P DAO1-in1m1 , showed very similar luciferase activity to the wild-type promoter upon induction with D-alanine. The inducible system was tunable by adjusting the levels of inducers, carbon source and nitrogen source. The intron 1-containing DAO1 promoters coupled with a DAO1 null

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

PubMed

Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

2016-01-01

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

SYSTEMIC BIOMARKERS AND CARDIAC GENE EXPRESSION PROFILES OF RAT DISEASE MODELS EMPLOYED IN AIR POLLUTION STUDIES

EPA Science Inventory

Cardiovascular disease (CVD) models are used for identification of mechanisms of susceptibility to air pollution. We hypothesized that baseline systemic biomarkers and cardiac gene expression in CVD rat models will have influence on their ozone-induced lung inflammation. Male 12-...

The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.

PubMed

Gawin, Agnieszka; Valla, Svein; Brautaset, Trygve

2017-07-01

The XylS/Pm regulator/promoter system originating from the Pseudomonas putida TOL plasmid pWW0 is widely used for regulated low- and high-level recombinant expression of genes and gene clusters in Escherichia coli and other bacteria. Induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose-dependent manner and are typically not metabolized by the host cells. Combinatorial mutagenesis and selection using the bla gene encoding β-lactamase as a reporter have demonstrated that the Pm promoter, the DNA sequence corresponding to the 5' untranslated end of its cognate mRNA and the xylS coding region can be modified and improved relative to various types of applications. By combining such mutant genetic elements, altered and extended expression profiles were achieved. Due to their unique properties, obtained systems serve as a genetic toolbox valuable for heterologous protein production and metabolic engineering, as well as for basic studies aiming at understanding fundamental parameters affecting bacterial gene expression. The approaches used to modify XylS/Pm should be adaptable for similar improvements also of other microbial expression systems. In this review, we summarize constructions, characteristics, refinements and applications of expression tools using the XylS/Pm system. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Expression of GAT1 in male reproductive system and its effects on reproduction in mice.

PubMed

Zhang, JinFu; Gui, YaPing; Yuan, Tao; Bian, CuiDong; Guo, LiHe

2009-12-01

The present study was carried out to identify GABA (gamma-aminobutyric acid) transport protein I (GAT1) in male reproductive organs and to study the effect of GAT1 overexpression on the male reproductive system in GAT1 transgenic mice (TG). Expression and location of GAT1 in testes, epididymis, and sperm of wild-type (WT) mice were identified by immunohistochemistry and western-blot. Histological changes of testes, epididymis, and sperm of transgenic mice overexpressing GAT1 were detected by immunofluorenscent staining and haematoxylin and eosin (HE) staining. GAT1 expression was detected in the testes, epididymis, and sperm of non-transgenic mice. Vacuolization and deformity of spermatogenic cells were observed in the transgenic mice, but the epididymis was unremarkable. Immunofluorenscent staining showed that the number of diastrophic and decapitated sperm increased significantly in transgenic mice to 46.9% from 7.3% in nontransgenic mice. These results suggest that abnormal expression of GAT1 could result in spermiogenesis function injury, sperm paramorphia and dysgenesis.

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.

Loss of Pgc-1α expression in aging mouse muscle potentiates glucose intolerance and systemic inflammation.

PubMed

Sczelecki, Sarah; Besse-Patin, Aurèle; Abboud, Alexandra; Kleiner, Sandra; Laznik-Bogoslavski, Dina; Wrann, Christiane D; Ruas, Jorge L; Haibe-Kains, Benjamin; Estall, Jennifer L

2014-01-15

Diabetes risk increases significantly with age and correlates with lower oxidative capacity in muscle. Decreased expression of peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc-1α) and target gene pathways involved in mitochondrial oxidative phosphorylation are associated with muscle insulin resistance, but a causative role has not been established. We sought to determine whether a decline in Pgc-1α and oxidative gene expression occurs during aging and potentiates the development of age-associated insulin resistance. Muscle-specific Pgc-1α knockout (MKO) mice and wild-type littermate controls were aged for 2 yr. Genetic signatures of skeletal muscle (microarray and mRNA expression) and metabolic profiles (glucose homeostasis, mitochondrial metabolism, body composition, lipids, and indirect calorimetry) of mice were compared at 3, 12, and 24 mo of age. Microarray and gene set enrichment analysis highlighted decreased function of the electron transport chain as characteristic of both aging muscle and loss of Pgc-1α expression. Despite significant reductions in oxidative gene expression and succinate dehydrogenase activity, young mice lacking Pgc-1α in muscle had lower fasting glucose and insulin. Consistent with loss of oxidative capacity during aging, Pgc-1α and Pgc-1β expression were reduced in aged wild-type mouse muscle. Interestingly, the combination of age and loss of muscle Pgc-1α expression impaired glucose tolerance and led to increased fat mass, insulin resistance, and inflammatory markers in white adipose and liver tissues. Therefore, loss of Pgc-1α expression and decreased mitochondrial oxidative capacity contribute to worsening glucose tolerance and chronic systemic inflammation associated with aging.

Regulation of melanopsin expression.

PubMed

Hannibal, Jens

2006-01-01

Circadian rhythms in mammals are adjusted daily to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Retinal ganglion cells (RGCs) of the RHT constitute a separate light-detecting system in the mammalian retina used for irradiance detection and for transmission to the circadian system and other non-imaging forming processes in the brain. The RGCs of the RHT are intrinsically photosensitive due to the expression of melanopsin, an opsin-like photopigment. This notion is based on anatomical and functional data and on studies of mice lacking melanopsin. Furthermore, heterologous expression of melanopsin in non-neuronal mammalian cell lines was found sufficient to render these cells photosensitive. Even though solid evidence regarding the function of melanopsin exists, little is known about the regulation of melanopsin gene expression. Studies in albino Wistar rats showed that the expression of melanopsin is diurnal at both the mRNA and protein levels. The diurnal changes in melanopsin expression seem, however, to be overridden by prolonged exposure to light or darkness. Significant increase in melanopsin expression was observed from the first day in constant darkness and the expression continued to increase during prolonged exposure in constant darkness. Prolonged exposure to constant light, on the other hand, decreased melanopsin expression to an almost undetectable level after 5 days of constant light. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 days. These observations show that dual mechanisms regulate melanopsin gene expression and that the intrinsic light-responsive RGCs in the albino Wistar rat adapt their expression of melanopsin to environmental light and darkness.

Online handwritten mathematical expression recognition

NASA Astrophysics Data System (ADS)

Büyükbayrak, Hakan; Yanikoglu, Berrin; Erçil, Aytül

2007-01-01

We describe a system for recognizing online, handwritten mathematical expressions. The system is designed with a user-interface for writing scientific articles, supporting the recognition of basic mathematical expressions as well as integrals, summations, matrices etc. A feed-forward neural network recognizes symbols which are assumed to be single-stroke and a recursive algorithm parses the expression by combining neural network output and the structure of the expression. Preliminary results show that writer-dependent recognition rates are very high (99.8%) while writer-independent symbol recognition rates are lower (75%). The interface associated with the proposed system integrates the built-in recognition capabilities of the Microsoft's Tablet PC API for recognizing textual input and supports conversion of hand-drawn figures into PNG format. This enables the user to enter text, mathematics and draw figures in a single interface. After recognition, all output is combined into one LATEX code and compiled into a PDF file.

Serotonin systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors and induce anorexia via a leptin-independent pathway in mice.

PubMed

Nonogaki, Katsunori; Ohba, Yukie; Sumii, Makiko; Oka, Yoshitomo

2008-07-18

NEFA/nucleobindin2 (NUCB2), a novel satiety molecule, is associated with leptin-independent melanocortin signaling in the central nervous system. Here, we show that systemic administration of m-chlorophenylpiperazine (mCPP), a serotonin 5-HT1B/2C receptor agonist, significantly increased the expression of hypothalamic NUCB2 in wild-type mice. The increases in hypothalamic NUCB2 expression induced by mCPP were attenuated in 5-HT2C receptor mutant mice. Systemic administration of mCPP suppressed food intake in db/db mice with leptin receptor mutation as well as lean control mice. On the other hand, the expression of hypothalamic NUCB2 and proopiomelanocortin (POMC) was significantly decreased in hyperphagic and non-obese 5-HT2C receptor mutants compared with age-matched wild-type mice. Interestingly, despite increased expression of hypothalamic POMC, hypothalamic NUCB2 expression was decreased in 5-HT2C receptor mutant mice with heterozygous mutation of beta-endorphin gene. These findings suggest that 5-HT systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors, and induce anorexia via a leptin-independent pathway in mice.

Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

PubMed Central

Sato, M; Figueiredo, ML; Burton, JB; Johnson, M; Chen, M; Powell, R; Gambhir, SS; Carey, M; Wu, L

2009-01-01

Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer. PMID:18305574

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

PubMed

Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

2000-02-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

PubMed Central

Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

2000-01-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

Comparative analysis of cadherin expression and connectivity patterns in the cerebellar system of ferret and mouse.

PubMed

Neudert, Franziska; Nuernberger, Krishna-K Monique; Redies, Christoph

2008-12-20

The cerebellum shows remarkable variations in the relative size of its divisions among vertebrate species. In the present study, we compare the cerebella of two mammals (ferret and mouse) by mapping the expression of three cadherins (cadherin-8, protocadherin-7, and protocadherin-10) at similar postnatal stages. The three cadherins are expressed differentially in parasagittal stripes in the cerebellar cortex, in the portions of the deep cerebellar nuclei, in the divisions of the inferior olivary nucleus, and in the lateral vestibular nucleus. The expression profiles suggest that the cadherin-positive structures are interconnected. The expression patterns resemble each other in ferret and mouse, although some differences can be observed. The general resemblance indicates that cerebellar organization is based on a common set of embryonic divisions in the two species. Consequently, the large differences in cerebellar morphology between the two species are more likely caused by differential growth of these embryonic divisions than by differences in early embryonic patterning. Based on the cadherin expression patterns, a model of corticonuclear projection territories in ferret and mouse is proposed. In summary, our results indicate that the cerebellar systems of rodents and carnivores display a relatively large degree of similarity in their molecular and functional organization.

[Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1 fusion gene].

PubMed

Luo, Dong-jiao; Yan, Jie; Mao, Ya-fei; Li, Shu-ping; Luo, Yi-hui; Li, Li-wei

2005-01-01

To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients. lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method. The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable. lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

USDA-ARS?s Scientific Manuscript database

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

Immunohistochemical expression of p53 and its clinicopathological correlation with modified Anneroth's histological grading system.

PubMed

Dave, Kajal V; Chalishazar, Monali; Dave, Vishal R; Panja, Pritam; Singh, Manisha; Modi, Tapan G

2016-01-01

Oral squamous cell carcinoma (OSCC) is an epithelial neoplasm generally beginning as focal overgrowth of altered stem cells near the basement membrane, moving upward and laterally, replacing the normal epithelium. Histopathological grading has been used for many decades in an attempt to predict the clinical behavior of oral squamous cell carcinoma. In the present study, Forty biopsies were studied for histological grading and p53 expression. The p53 expression was studied in relation to clinical parameters such as age, sex of patient and site of tumors. Relation between histological grade of malignancy and p53 protein expression was analysed. All cases were classified according to Anneroth's histological malignancy grading system (1987). 40 cases of OSCC were assessed for clinical parameters, Anneroth's histological grading and immunohistochemically stained with p53 protien. The results obtained were analyzed using Spearman's Co-relation. The positive expression of p53 was found in 62% of carcinomas studied. Positivity of p53 showed correlation with histological grade of malignancy and with individual parameters like degree of keratinization, nuclear polymorphism, number of mitoses and lymphoplasmacytic infiltration while showed a negative correlation with pattern of invasion. Our study showed a significant correlation between parameters of tumor cell population, lymphoplasmacytic infiltration and p53 expression. A significant association between high grade of malignancy and p53 overexpression and insignificant correlation of p53 with age, sex of the patient and site of the tumor was found.

Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli.

PubMed

Chiang, Chung-Jen; Chen, Hong-Chen; Chao, Yun-Peng; Tzen, Jason T C

2005-06-15

Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites

NASA Technical Reports Server (NTRS)

Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

2003-01-01

This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

Moon Express Media Event

NASA Image and Video Library

2014-11-03

Rob Mueller, NASA senior technologist in the Surface Systems Office in Kennedy Space Center's Engineering and Technology Directorate, demonstrates the Regolith Advanced Surface System Operations Robot, or RASSOR, during a media event at Kennedy's automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility. The event was held to announce Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST.

Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes.

PubMed

Chen, Yuan; Ding, Yun; Zhang, Zuming; Wang, Wen; Chen, Jun-Yuan; Ueno, Naoto; Mao, Bingyu

2011-12-20

The evolution of the central nervous system (CNS) is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties, gene duplication might play an important role in the functional innovation of vertebrate CNS. In this study, we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution. We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri. Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos, and more than 50% and 30% duplicate genes are expressed in the telencephalon and mid-hindbrain boundary, respectively, which are mostly considered as two innovations in the vertebrate CNS. Interestingly, more than 50% of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization, indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates. Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes. Copyright © 2011. Published by Elsevier Ltd.

Use of baculovirus expression system for generation of virus-like particles: successes and challenges.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

2013-08-01

The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression of eukaryotic polypeptides in chloroplasts

DOEpatents

Mayfield, Stephen P.

2013-06-04

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

Prevention of pathology in mdx mice by expression of utrophin: analysis using an inducible transgenic expression system.

PubMed

Squire, S; Raymackers, J M; Vandebrouck, C; Potter, A; Tinsley, J; Fisher, R; Gillis, J M; Davies, K E

2002-12-15

Duchenne muscular dystrophy results from the absence of dystrophin, a cytoskeletal protein. Previously, we have shown in a transgenic mouse model of the disease (mdx) that high levels of expression of the dystrophin-related protein, utrophin can prevent pathology. We developed a new transgenic mouse model where muscle specific utrophin expression was conditioned by addition of tetracycline in water. Transgene expression was turned on at different time points: in utero, at birth, 10 and 30 days after birth. We obtained moderate levels of expression, variable from fibre to fibre (mosaicism) but sufficient to induce a correct localization of the dystro-sarcoglycan complex. Histology revealed a reduction of necrotic foci and of the percentage of centronucleated fibres, which remained still largely above the normal level. Isometric force was not improved but the resistance to eccentric contractions was significantly stronger. When utrophin expression was activated 30 days after birth, improvements were marginal, suggesting that the age at which utrophin therapy is initiated could be an important factor. Our results also provide an unexpected insight into the pathogenesis of the dystrophinopathies. We observed a complete normalization of the characteristics of the mechano-sensitive/voltage-independent Ca(2+) channels (occurrence, open probabilities and Ca(2+) currents), while the classical markers of dystrophy were still abnormal. These observations question the role of increased Ca(2+) channel activity in initiating the dystrophic process. The new model shows that utrophin therapy, initiated after birth, can be effective, but the extent of correction of the various symptoms of dystrophinopathy critically depends on the amount of utrophin expressed.

Burkholderia mallei and Burkholderia pseudomallei Cluster 1 Type VI Secretion System Gene Expression Is Negatively Regulated by Iron and Zinc

PubMed Central

Burtnick, Mary N.; Brett, Paul J.

2013-01-01

Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc. PMID:24146925

Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

PubMed

Burtnick, Mary N; Brett, Paul J

2013-01-01

Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM) were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

PubMed

Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

2015-11-01

Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

[Tissue localization and expression difference of endogenous beta-glucosidase in digestive system of Musca domestica third instar larvae].

PubMed

Hu, Rong; Zhang, Shu; Wu, Jian-Wei; Guo, Guo; Fu, Ping

2013-08-01

To study the tissue localization and expression difference of endogenous beta-glucosidase in digestive system of Musca domestica third instar larvae. The digestive system of the 3rd instar larvae of Musca domestic was taken for the below tests. Tissue localization of endogenous beta-glucosidase mRNA was identified by in situ hybridization. Cellulase was localized by immunohistochemistry. The enzymatic activity of beta-glucosidase was measured by 3, 5-dinitrosalicylic acid(DNS) assay. The relative mRNA expression levels of M. domestica beta-glucosidase gene in these organs were determined by RT-PCR. Beta-glucosidase mRNA, with in situ hybridization, was shown in the epithelial cells of midgut, salivary glands and foregut of the larvae. The immunohistochemical analysis on larvae tissues revealed that cellulase was produced and secreted by the epithelial cells of the midgut, salivary glands and foregut. beta-glucosidase activity in salivary glands, foregut, midgut, and hindgut was (0.80 +/- 0.06), (0.38 +/- 0.02), (1.20 +/- 0.05) and (0.26 +/- 0.02) IU/mg, respectively. There was significant difference in beta-glucosidase activity among these digestive organs (P < 0.05). The activity level of beta-glucosidase was highest in midgut [(45.45 +/- 1.27)%], and lowest in hindgut [(9.85 +/- 0.88)%]. However, beta-glucosidase gene were only expressed in the salivary gland, foregut and midgut. Significant differences in gene expression level of beta-glucosidase was found among these organs (P < 0.05). The relative expression quantity of beta-glucosidase gene in midgut and salivary glands were 5 and 3 times higher than that in foregut. The endogenous beta-glucosidase gene is expressed in the foregut, midgut and salivary glands. The midgut and salivary glands of Musca domestica 3rd instar larvae are the primary organs of this enzyme secretion.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

PubMed

Shamekova, Malika; Mendoza, Maria R; Hsieh, Yi-Cheng; Lindbo, John; Omarov, Rustem T; Scholthof, Herman B

2014-03-01

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing. Copyright © 2014. Published by Elsevier Inc.

Expression of the System N transporter (SNAT5/SN2) during development indicates its plausible role in glutamatergic neurotransmission.

PubMed

Rodríguez, Angelina; Ortega, Arturo; Berumen, Laura C; García-Alcocer, María G; Giménez, Cecilio; Zafra, Francisco

2014-07-01

Solute neutral amino acid transporter 5 (SNAT5/SN2) is a member of the System N family, expressed in glial cells in the adult brain, able to transport glutamine, histidine or glycine among other substrates. Its tight association with synapses and its electroneutral mode of operation that allows the bidirectional movement of substrates, supports the idea that this transporter participates in the function of the glutamine-glutamate cycle between neurons and glia. Moreover, SNAT5/SN2 might contribute to the regulation of glycine concentration in glutamatergic synapses and, therefore, to the functioning of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors. Ontogenic maturation of these synapses occurs postnatally through the coordinate expression of a large number of receptors, transporters, structural and regulatory proteins that ensure the correct operation of the excitatory pathways in the central nervous system. Since the temporal pattern of expression of SNAT5/SN2 is unknown, we analyzed it by immunoblot and immunohistochemical techniques. Results indicate that the expression of SNAT5/SN2 is triggered between the second and third postnatal week in the cerebral cortex, in parallel to the expression of the vesicular glutamate transporter vGLUT1 and the glial glutamate transporter GLT1/EAAT2. In the cerebellum, this process occurs about one week later than in the cerebral cortex. Immunohistochemical staining of cortical sections shows that from postnatal day 14 to adulthood the transporter was expressed exclusively in glial cells. Our results are consistent with the idea that SNAT5/SN2 expression is coordinated with that of other proteins necessary for the operation of glutamatergic synapses and reinforce the existence of a regulatory cross-talk between neurons and glia that orchestrates the building up of these synapses. Copyright © 2014 Elsevier Ltd. All rights reserved.

A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor.

PubMed

Poon, S K; Peacock, L; Gibson, W; Gull, K; Kelly, S

2012-02-01

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.

A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor

PubMed Central

Poon, S. K.; Peacock, L.; Gibson, W.; Gull, K.; Kelly, S.

2012-01-01

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes. PMID:22645659

Real-Time Characterization of Virulence Factor Expression in Yersinia pestis Using a Green Fluorescent Protein Reporter System

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

Forde, C; Rocco, J; Fitch, J P

2004-06-09

A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopK, yopT, yscN, and lcrE/yopN, which are all components of the Type III secretion virulence mechanism of Y. pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time. Basal expression levels observed for the Y. pestis promoters, expressedmore » as percentages of the positive control with GFP under the control of the lac promoter, were: yopE (15%), sycE (15%), yopK (13%), yopT (4%), lcrE (3.3%) and yscN (0.8%). The yopE reporter showed the strongest gene induction following temperature transition from 26 C to 37 C. The induction levels of the other virulence factors, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11 fold), yscN (7 fold), yopK (6 fold), lcrE (3 fold), yopT (2 fold), and sycE (2 fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y. pestis virulence factors in real time, in living cells.« less

A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level

PubMed Central

Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

2015-01-01

Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum. PMID:25488698

SOBP Is Mutated in Syndromic and Nonsyndromic Intellectual Disability and Is Highly Expressed in the Brain Limbic System

PubMed Central

Birk, Efrat; Har-Zahav, Adi; Manzini, Chiara M.; Pasmanik-Chor, Metsada; Kornreich, Liora; Walsh, Christopher A.; Noben-Trauth, Konrad; Albin, Adi; Simon, Amos J.; Colleaux, Laurence; Morad, Yair; Rainshtein, Limor; Tischfield, David J.; Wang, Peter; Magal, Nurit; Maya, Idit; Shoshani, Noa; Rechavi, Gideon; Gothelf, Doron; Maydan, Gal; Shohat, Mordechai; Basel-Vanagaite, Lina

2010-01-01

Intellectual disability (ID) affects 1%–3% of the general population. We recently reported on a family with autosomal-recessive mental retardation with anterior maxillary protrusion and strabismus (MRAMS) syndrome. One of the reported patients with ID did not have dysmorphic features but did have temporal lobe epilepsy and psychosis. We report on the identification of a truncating mutation in the SOBP that is responsible for causing both syndromic and nonsyndromic ID in the same family. The protein encoded by the SOBP, sine oculis binding protein ortholog, is a nuclear zinc finger protein. In mice, Sobp (also known as Jxc1) is critical for patterning of the organ of Corti; one of our patients has a subclinical cochlear hearing loss but no gross cochlear abnormalities. In situ RNA expression studies in postnatal mouse brain showed strong expression in the limbic system at the time interval of active synaptogenesis. The limbic system regulates learning, memory, and affective behavior, but limbic circuitry expression of other genes mutated in ID is unusual. By comparing the protein content of the +/jc to jc/jc mice brains with the use of proteomics, we detected 24 proteins with greater than 1.5-fold differences in expression, including two interacting proteins, dynamin and pacsin1. This study shows mutated SOBP involvement in syndromic and nonsyndromic ID with psychosis in humans. PMID:21035105

Serotonin 1A receptor (5-HT1A) of the sea lamprey: cDNA cloning and expression in the central nervous system.

PubMed

Cornide-Petronio, María Eugenia; Anadón, Ramón; Barreiro-Iglesias, Antón; Rodicio, María Celina

2013-09-01

Serotonergic cells are among the earliest neurons to be born in the developing central nervous system and serotonin is known to regulate the development of the nervous system. One of the major targets of the activity of serotonergic cells is the serotonin 1A receptor (5-HT1A), an ancestral archetypical serotonin receptor. In this study, we cloned and characterized the 3D structure of the sea lamprey 5-HT1A, and studied the expression of its transcript in the central nervous system by means of in situ hybridization. In phylogenetic analyses, the sea lamprey 5-HT1A sequence clustered together with 5-HT1A sequences of vertebrates and emerged as an outgroup to all gnathostome sequences. In situ hybridization analysis during prolarval, larval and adult stages showed a widespread expression of the lamprey 5-ht1a transcript. In P1 prolarvae 5-ht1a mRNA expression was observed in diencephalic nuclei, the rhombencephalon and rostral spinal cord. At P2 prolarval stage the 5-ht1a expression extended to other brain areas including telencephalic regions. 5-ht1a expression in larvae was observed throughout almost all the main brain regions with the strongest expression in the olfactory bulbs, lateral pallium, striatum, preoptic region, habenula, prethalamus, thalamus, pretectum, hypothalamus, rhombencephalic reticular area, dorsal column nucleus and rostral spinal cord. In adults, the 5-ht1a transcript was also observed in cells of the subcommissural organ. Comparison of the expression of 5-ht1a between the sea lamprey and other vertebrates reveals a conserved pattern in most of the brain regions, likely reflecting the ancestral vertebrate condition.

Divergent Annexin A1 expression in periphery and gut is associated with systemic immune activation and impaired gut immune response during SIV infection

PubMed Central

Sena, Angela A. S.; Glavan, Tiffany; Jiang, Guochun; Sankaran-Walters, Sumathi; Grishina, Irina; Dandekar, Satya; Goulart, Luiz R.

2016-01-01

HIV-1 disease progression is paradoxically characterized by systemic chronic immune activation and gut mucosal immune dysfunction, which is not fully defined. Annexin A1 (ANXA1), an inflammation modulator, is a potential link between systemic inflammation and gut immune dysfunction during the simian immunodeficiency virus (SIV) infection. Gene expression of ANXA1 and cytokines were assessed in therapy-naïve rhesus macaques during early and chronic stages of SIV infection and compared with SIV-negative controls. ANXA1 expression was suppressed in the gut but systemically increased during early infection. Conversely, ANXA1 expression increased in both compartments during chronic infection. ANXA1 expression in peripheral blood was positively correlated with HLA-DR+CD4+ and CD8+ T-cell frequencies, and negatively associated with the expression of pro-inflammatory cytokines and CCR5. In contrast, the gut mucosa presented an anergic cytokine profile in relation to ANXA1 expression. In vitro stimulations with ANXA1 peptide resulted in decreased inflammatory response in PBMC but increased activation of gut lymphocytes. Our findings suggest that ANXA1 signaling is dysfunctional in SIV infection, and may contribute to chronic inflammation in periphery and with immune dysfunction in the gut mucosa. Thus, ANXA1 signaling may be a novel therapeutic target for the resolution of immune dysfunction in HIV infection. PMID:27484833

Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins

PubMed Central

2018-01-01

ABSTRACT Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (Pf) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the Pf of the oocytes. In fact, the Pf values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1FLAG [FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys – hAQP1C189S mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 – were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes. PMID

Apelin-APJ system is responsible for stress-induced increase in atrial natriuretic peptide expression in rat heart.

PubMed

Izgut-Uysal, Vecihe Nimet; Acar, Nuray; Birsen, Ilknur; Ozcan, Filiz; Ozbey, Ozlem; Soylu, Hakan; Avci, Sema; Tepekoy, Filiz; Akkoyunlu, Gokhan; Yucel, Gultekin; Ustunel, Ismail

2018-04-01

The cardiovascular system is a primary target of stress and stress is the most important etiologic factor in cardiovascular diseases. Stressors increase expressions of atrial natriuretic peptide (ANP) and apelin in cardiac tissue. The aim of the present study was to investigate whether stress-induced apelin has an effect on the expression of ANP in the right atrium of rat heart. The rats were divided into the control, stress and F13A+stress groups. In the stress and F13A+stress groups, the rats were subjected to water immersion and restraint stress (WIRS) for 6h. In the F13A+stress group, apelin receptor antagonist F13A, was injected intravenously immediately before application of WIRS. The plasma samples were obtained for the measurement of corticosterone and atrial natriuretic peptide. The atrial samples were used for immunohistochemistry and western blot analysis. F13A administration prevented the rise of plasma corticosterone and ANP levels induced by WIRS. While WIRS application increased the expressions of apelin, HIF-1α and ANP in atrial tissue, while F13A prevented the stress-induced increase in the expression of HIF-1α and ANP. Stress-induced apelin induces ANP expression in atrial tissue and may play a role in cardiovascular homeostasis by increasing ANP expression under WIRS conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aqueous synthesis of highly luminescent AgInS2-ZnS quantum dots and their biological applications

NASA Astrophysics Data System (ADS)

Regulacio, Michelle D.; Win, Khin Yin; Lo, Seong Loong; Zhang, Shuang-Yuan; Zhang, Xinhai; Wang, Shu; Han, Ming-Yong; Zheng, Yuangang

2013-02-01

Highly emissive and air-stable AgInS2-ZnS quantum dots (ZAIS QDs) with quantum yields of up to 20% have been successfully synthesized directly in aqueous media in the presence of polyacrylic acid (PAA) and mercaptoacetic acid (MAA) as stabilizing and reactivity-controlling agents. The as-prepared water-dispersible ZAIS QDs are around 3 nm in size, possess the tetragonal chalcopyrite crystal structure, and exhibit long fluorescence lifetimes (>100 ns). In addition, these ZAIS QDs are found to exhibit excellent optical and colloidal stability in physiologically relevant pH values as well as very low cytotoxicity, which render them particularly suitable for biological applications. Their potential use in biological labelling of baculoviral vectors is demonstrated.Highly emissive and air-stable AgInS2-ZnS quantum dots (ZAIS QDs) with quantum yields of up to 20% have been successfully synthesized directly in aqueous media in the presence of polyacrylic acid (PAA) and mercaptoacetic acid (MAA) as stabilizing and reactivity-controlling agents. The as-prepared water-dispersible ZAIS QDs are around 3 nm in size, possess the tetragonal chalcopyrite crystal structure, and exhibit long fluorescence lifetimes (>100 ns). In addition, these ZAIS QDs are found to exhibit excellent optical and colloidal stability in physiologically relevant pH values as well as very low cytotoxicity, which render them particularly suitable for biological applications. Their potential use in biological labelling of baculoviral vectors is demonstrated. Electronic supplementary information (ESI) available: Quantum yields, EDX spectrum and photoluminescence decay curves. See DOI: 10.1039/c3nr34159c

A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus.

PubMed

Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang

2016-01-18

Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate's protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development.

A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus

PubMed Central

Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang

2016-01-01

Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate’s protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development. PMID:26777545

DNA microarray analysis identifies CKS2 and LEPR as potential markers of meningioma recurrence.

PubMed

Menghi, Francesca; Orzan, Francesca N; Eoli, Marica; Farinotti, Mariangela; Maderna, Emanuela; Pisati, Federica; Bianchessi, Donatella; Valletta, Lorella; Lodrini, Sandro; Galli, Giuseppe; Anghileri, Elena; Pellegatta, Serena; Pollo, Bianca; Finocchiaro, Gaetano

2011-01-01

Meningiomas are the most frequent intracranial tumors. Surgery can be curative, but recurrences are possible. We performed gene expression analyses and loss of heterozygosity (LOH) studies looking for new markers predicting the recurrence risk. We analyzed expression profiles of 23 meningiomas (10 grade I, 10 grade II, and 3 grade III) and validated the data using quantitative polymerase chain reaction (qPCR). We performed LOH analysis on 40 meningiomas, investigating chromosomal regions on 1p, 9p, 10q, 14q, and 22q. We found 233 and 268 probe sets to be significantly down- and upregulated, respectively, in grade II or III meningiomas. Genes downregulated in high-grade meningiomas were overrepresented on chromosomes 1, 6, 9, 10, and 14. Based on functional enrichment analysis, we selected LIM domain and actin binding 1 (LIMA1), tissue inhibitor of metalloproteinases 3 (TIMP3), cyclin-dependent kinases regulatory subunit 2 (CKS2), leptin receptor (LEPR), and baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) for validation using qPCR and confirmed their differential expression in the two groups of tumors. We calculated ΔCt values of CKS2 and LEPR and found that their differential expression (C-L index) was significantly higher in grade I than in grade II or III meningiomas (p < .0001). Interestingly, the C-L index of nine grade I meningiomas from patients who relapsed in <5 years was significantly lower than in grade I meningiomas from patients who did not relapse. These findings indicate that the C-L index may be relevant to define the progression risk in meningioma patients, helping guide their clinical management. A prospective analysis on a larger number of cases is warranted.

Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System

PubMed Central

Rahpeyma, Mehdi; Fotouhi, Fatemeh; Makvandi, Manouchehr; Ghadiri, Ata; Samarbaf-Zadeh, Alireza

2015-01-01

Background Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development. Objectives The current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system. Materials and Methods To express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system. Results Primary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter. Conclusions Characterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV. PMID:26862379

Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System.

PubMed

Rahpeyma, Mehdi; Fotouhi, Fatemeh; Makvandi, Manouchehr; Ghadiri, Ata; Samarbaf-Zadeh, Alireza

2015-11-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development. The current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system. To express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system. Primary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter. Characterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV.

Two-Component Signal Transduction Systems That Regulate the Temporal and Spatial Expression of Myxococcus xanthus Sporulation Genes.

PubMed

Sarwar, Zaara; Garza, Anthony G

2016-02-01

When starved for nutrients, Myxococcus xanthus produces a biofilm that contains a mat of rod-shaped cells, known as peripheral rods, and aerial structures called fruiting bodies, which house thousands of dormant and stress-resistant spherical spores. Because rod-shaped cells differentiate into spherical, stress-resistant spores and spore differentiation occurs only in nascent fruiting bodies, many genes and multiple levels of regulation are required. Over the past 2 decades, many regulators of the temporal and spatial expression of M. xanthus sporulation genes have been uncovered. Of these sporulation gene regulators, two-component signal transduction circuits, which typically contain a histidine kinase sensor protein and a transcriptional regulator known as response regulator, are among the best characterized. In this review, we discuss prototypical two-component systems (Nla6S/Nla6 and Nla28S/Nla28) that regulate an early, preaggregation phase of sporulation gene expression during fruiting body development. We also discuss orphan response regulators (ActB and FruA) that regulate a later phase of sporulation gene expression, which begins during the aggregation stage of fruiting body development. In addition, we summarize the research on a complex two-component system (Esp) that is important for the spatial regulation of sporulation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A distal modular enhancer complex acts to control pituitary- and nervous system-specific expression of the LHX3 regulatory gene.

PubMed

Mullen, Rachel D; Park, Soyoung; Rhodes, Simon J

2012-02-01

Lin-11, Isl-1, and Mec-3 (LIM)-homeodomain (HD)-class transcription factors are critical for many aspects of mammalian organogenesis. Of these, LHX3 is essential for pituitary gland and nervous system development. Pediatric patients with mutations in coding regions of the LHX3 gene have complex syndromes, including combined pituitary hormone deficiency and nervous system defects resulting in symptoms such as dwarfism, thyroid insufficiency, infertility, and developmental delay. The pathways underlying early pituitary development are poorly understood, and the mechanisms by which the LHX3 gene is regulated in vivo are not known. Using bioinformatic and transgenic mouse approaches, we show that multiple conserved enhancers downstream of the human LHX3 gene direct expression to the developing pituitary and spinal cord in a pattern consistent with endogenous LHX3 expression. Several transferable cis elements can individually guide nervous system expression. However, a single 180-bp minimal enhancer is sufficient to confer specific expression in the developing pituitary. Within this sequence, tandem binding sites recognized by the islet-1 (ISL1) LIM-HD protein are essential for enhancer activity in the pituitary and spine, and a pituitary homeobox 1 (PITX1) bicoid class HD element is required for spatial patterning in the developing pituitary. This study establishes ISL1 as a novel transcriptional regulator of LHX3 and describes a potential mechanism for regulation by PITX1. Moreover, these studies suggest models for analyses of the transcriptional pathways coordinating the expression of other LIM-HD genes and provide tools for the molecular analysis and genetic counseling of pediatric patients with combined pituitary hormone deficiency.

A Distal Modular Enhancer Complex Acts to Control Pituitary- and Nervous System-Specific Expression of the LHX3 Regulatory Gene

PubMed Central

Mullen, Rachel D.; Park, Soyoung

2012-01-01

Lin-11, Isl-1, and Mec-3 (LIM)-homeodomain (HD)-class transcription factors are critical for many aspects of mammalian organogenesis. Of these, LHX3 is essential for pituitary gland and nervous system development. Pediatric patients with mutations in coding regions of the LHX3 gene have complex syndromes, including combined pituitary hormone deficiency and nervous system defects resulting in symptoms such as dwarfism, thyroid insufficiency, infertility, and developmental delay. The pathways underlying early pituitary development are poorly understood, and the mechanisms by which the LHX3 gene is regulated in vivo are not known. Using bioinformatic and transgenic mouse approaches, we show that multiple conserved enhancers downstream of the human LHX3 gene direct expression to the developing pituitary and spinal cord in a pattern consistent with endogenous LHX3 expression. Several transferable cis elements can individually guide nervous system expression. However, a single 180-bp minimal enhancer is sufficient to confer specific expression in the developing pituitary. Within this sequence, tandem binding sites recognized by the islet-1 (ISL1) LIM-HD protein are essential for enhancer activity in the pituitary and spine, and a pituitary homeobox 1 (PITX1) bicoid class HD element is required for spatial patterning in the developing pituitary. This study establishes ISL1 as a novel transcriptional regulator of LHX3 and describes a potential mechanism for regulation by PITX1. Moreover, these studies suggest models for analyses of the transcriptional pathways coordinating the expression of other LIM-HD genes and provide tools for the molecular analysis and genetic counseling of pediatric patients with combined pituitary hormone deficiency. PMID:22194342

Ovariectomy modify local renin-angiotensin-aldosterone system gene expressions in the heart of ApoE (-/-) mice.

PubMed

Borges, Celina Carvalho; Penna-de-Carvalho, Aline; Medeiros Junior, Jorge L; Aguila, Marcia Barbosa; Mandarim-de-Lacerda, Carlos A

2017-12-15

The evaluation of the local Renin-Angiotensin-Aldosterone system (RAAS) gene expressions in the heart of ovariectomized (OVX) apolipoprotein E deficient mice (ApoE). Four-months old C57BL/6 female mice (wild-type, wt, n=20), and ApoE female mice (n=20), were submitted to OVX or a surgical procedure without ovary removal (SHAM) and formed four groups (n=10/group): SHAM/wt, SHAM/ApoE, OVX/wt, and OVX/ApoE. OVX led to greater body mass, plasma triglycerides (TG) and total cholesterol, and resulted in insulin resistance and altered RAAS gene expressions in the heart tissue. The gene expression of angiotensin-converting enzyme (ACE)-2 was lower in OVX/wt than in SHAM/wt (P=0.0004), Mas receptor (MASr) was lower in OVX/wt compared to SHAM/wt (P<0.0001). Also, angiotensin II receptor type 1 (AT1r) was higher in OVX/wt than in SHAM/wt (P=0.0229), and AT2r was lower in OVX/wt than in SHAM/wt (P=0.0121). OVX and ApoE deficiency showed interaction potentializing the insulin resistance, increasing TG levels and altering ACE and MASr gene expressions. ACE gene expression was higher in OVX/ApoE than in OVX/wt (P<0.0001), and MASr gene expression was lower in OVX/ApoE than in OVX/wt (P<0.0001). The impact of OVX on local RAAS cascade in the heart of ApoE deficient animals, besides the metabolic changes culminating with insulin resistance, involves an upregulation of renin, ACE, and AT1r gene expressions. The findings may contribute to clarify the mechanisms of development of postmenopausal hypertension and the link between RAAS and apolipoprotein E. Copyright © 2017 Elsevier Inc. All rights reserved.

The two-component system GrvRS (EtaRS) regulates ace expression in Enterococcus faecalis OG1RF.

PubMed

Roh, Jung Hyeob; Singh, Kavindra V; La Rosa, Sabina Leanti; Cohen, Ana Luisa V; Murray, Barbara E

2015-01-01

Expression of ace (adhesin to collagen of Enterococcus faecalis), encoding a virulence factor in endocarditis and urinary tract infection models, has been shown to increase under certain conditions, such as in the presence of serum, bile salts, urine, and collagen and at 46 °C. However, the mechanism of ace/Ace regulation under different conditions is still unknown. In this study, we identified a two-component regulatory system GrvRS as the main regulator of ace expression under these stress conditions. Using Northern hybridization and β-galactosidase assays of an ace promoter-lacZ fusion, we found transcription of ace to be virtually absent in a grvR deletion mutant under the conditions that increase ace expression in wild-type OG1RF and in the complemented strain. Moreover, a grvR mutant revealed decreased collagen binding and biofilm formation as well as attenuation in a murine urinary tract infection model. Here we show that GrvR plays a major role in control of ace expression and E. faecalis virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Moon Express Media Event

NASA Image and Video Library

2014-11-03

Rob Mueller, left, NASA senior technologist in the Surface Systems Office in Kennedy Space Center's Engineering and Technology Directorate, talks with former NASA Apollo astronaut Buzz Aldrin during a demonstration of the Regolith Advanced Surface Systems Operations Robot, or RASSOR, at the automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida. The event was held to announce Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST.

Moon Express Media Event

NASA Image and Video Library

2014-11-03

Rob Mueller, left, NASA senior technologist in the Surface Systems Office in Kennedy Space Center's Engineering and Technology Directorate, talks with former NASA Apollo astronaut Buzz Aldrin during a demonstration of the Regolith Advanced Surface System Operations Robot, or RASSOR, at the automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida. The event was held to announce Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST.

Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems

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

Conover, David R.; Crawford, Alasdair J.; Fuller, Jason

This Protocol provides a set of “best practices” for characterizing energy storage systems (ESSs) and measuring and reporting their performance. It serves as a basis for assessing how an ESS will perform with respect to key performance attributes relevant to different applications. It is intended to provide a valid and accurate basis for the comparison of different ESSs. By achieving the stated purpose, the Protocol will enable more informed decision-making in the selection of ESSs for various stationary applications. The Protocol identifies general information and technical specifications relevant in describing an ESS and also defines a set of test, measurement,more » and evaluation criteria with which to express the performance of ESSs that are intended for energy-intensive and/or power-intensive stationary applications. An ESS includes a storage device, battery management system, and any power conversion systems installed with the storage device. The Protocol is agnostic with respect to the storage technology and the size and rating of the ESS. The Protocol does not apply to single-use storage devices and storage devices that are not coupled with power conversion systems, nor does it address safety, security, or operations and maintenance of ESSs, or provide any pass/fail criteria.« less

Expressions for the precession quantities based upon the IAU /1976/ system of astronomical constants

NASA Technical Reports Server (NTRS)

Lieske, J. H.; Lederle, T.; Fricke, W.; Morando, B.

1977-01-01

The structure of the expressions usually employed in calculating the effects of precession is examined, and a method is outlined for revising the expressions to account for changes in the fundamental astronomical constants. It is shown that the basic set of parameters, upon which depend the lengthy polynomials for computing the mean obliquity of data and the elements of the precession matrix, consists of the mean obliquity, the speed of general precession in longitude at a fixed epoch, and the system of planetary masses. Special attention is given to the motion of the ecliptic pole, formulations for a basic epoch as well as an arbitrary epoch, and ecliptic motion relative to the basic epoch. Numerical precession quantities at epoch J2000.0 (JED 2451545.0) are presented which result from the revision of astronomical constants adopted at the XVI General Assembly of the IAU.

The timing and location of GDNF expression determines enteric nervous system structure and function

PubMed Central

Wang, Hongtao; Hughes, Inna; Planer, William; Parsadanian, Alexander; Grider, John R.; Vohra, Bhupinder P.S.; Keller-Peck, Cynthia; Heuckeroth, Robert O.