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

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

Selection of reference genes for expression studies with fish myogenic cell cultures.

PubMed

Bower, Neil I; Johnston, Ian A

2009-08-10

Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. The geometric average of any three of Hprt1, Ef1alpha, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

PubMed

Thyagarajan, Bhaskar; Scheyhing, Kelly; Xue, Haipeng; Fontes, Andrew; Chesnut, Jon; Rao, Mahendra; Lakshmipathy, Uma

2009-03-01

Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

PubMed Central

Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

2013-01-01

Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

Targeting BRCAness in Gastric Cancer

DTIC Science & Technology

2017-10-01

modified CRISPR system using dCas9-KRAB expressing variants of these cells, and validated them for CRISPRi screening. These reagents will next be used...OE19 Adherent Stable Oesophagus/ gastric cardia - - - OE33 Adherent Stable Esophageal adenocarcinoma - - - Figure 2. Validation of CRISPR ...project planning and reporting Patrick O’Leary Postdoctoral Fellow UCSF PH.D. Design, execution, and interpretation of CRISPR experiments

Stable Nuclear Transformation System for the Coccolithophorid Alga Pleurochrysis carterae

PubMed Central

Endo, Hirotoshi; Yoshida, Megumi; Uji, Toshiki; Saga, Naotsune; Inoue, Koji; Nagasawa, Hiromichi

2016-01-01

Of the three dominant marine microalgal groups, dinoflagellates and diatoms can undergo genetic transformation; however, no transformation method has been established for haptophytes to date. Here, we report the first stable genetic transformation of a coccolithophore, Pleurochrysis carterae, by means of polyethylene glycol (PEG)-mediated transfer of a bacterial hygromycin B-resistance gene. Together with the novel transient green fluorescent protein (GFP) expression system, this approach should facilitate further molecular-based research in this phylum. PMID:26947136

Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

USDA-ARS?s Scientific Manuscript database

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

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

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

PubMed Central

Szczesny, Roman J.; Kowalska, Katarzyna; Klosowska-Kosicka, Kamila; Chlebowski, Aleksander; Owczarek, Ewelina P.; Warkocki, Zbigniew; Kulinski, Tomasz M.; Adamska, Dorota; Affek, Kamila; Jedroszkowiak, Agata; Kotrys, Anna V.; Tomecki, Rafal; Krawczyk, Pawel S.; Borowski, Lukasz S.; Dziembowski, Andrzej

2018-01-01

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq. PMID:29590189

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

PubMed

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

2012-12-01

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

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction

PubMed Central

Goldfless, Stephen J.; Wagner, Jeffrey C.; Niles, Jacquin C.

2014-01-01

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum. PMID:25370483

Social instability and immunity in rhesus monkeys: the role of the sympathetic nervous system.

PubMed

Capitanio, John P; Cole, Steven W

2015-05-26

Social instability can adversely affect endocrine, immune and health outcomes, and recent evidence suggests that the sympathetic nervous system (SNS) might mediate these effects. We conducted two studies with adult male rhesus monkeys (Macaca mulatta) to understand how social conditions affect measures of SNS activity and immune function. In Experiment 1, animals were socialized in stable social conditions, then were switched to unstable (stressful) social conditions, then were returned to stable conditions. Analysis revealed quadratic effects for measures of behaviour, urinary metabolites of epinephrine and norepinephrine, and expression of immune response genes: as expected, social instability adversely impacted most measures, and the effects remediated upon re-imposition of stable conditions. Cortisol levels were unaffected. In Experiment 2, we used the sympathomimetic drug methamphetamine to challenge the SNS; animals also underwent socialization in stable or unstable groups. Surprisingly, while methamphetamine elevated plasma catecholamines, responses in lymph nodes tracked the social, and not the drug, condition: social instability upregulated the density of SNS fibres in lymph nodes and downregulated Type I interferon gene expression. Together, these results indicate that the SNS is extremely sensitive to social conditions; full understanding of the adverse effects of social instability on health should therefore incorporate measures of this health-relevant system. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines.

PubMed

Wu, Yong; Zhang, Tian-Ying; Fang, Lin-Lin; Chen, Zi-Xuan; Song, Liu-Wei; Cao, Jia-Li; Yang, Lin; Yuan, Quan; Xia, Ning-Shao

2016-08-01

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study. Copyright © 2016. Published by Elsevier B.V.

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

PubMed

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

2015-02-01

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

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

PubMed Central

Wang, Yarong; Du, Dewei; Li, Zhanting; Wei, Junxia; Yang, Angang

2012-01-01

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats. PMID:22990115

Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon.

PubMed

Howe, Kevin; Karsi, Attila; Germon, Pierre; Wills, Robert W; Lawrence, Mark L; Bailey, Richard H

2010-07-23

Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum. We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies. This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.

Mechanisms of stable lipid loss in a social insect

PubMed Central

Ament, Seth A.; Chan, Queenie W.; Wheeler, Marsha M.; Nixon, Scott E.; Johnson, S. Peir; Rodriguez-Zas, Sandra L.; Foster, Leonard J.; Robinson, Gene E.

2011-01-01

SUMMARY Worker honey bees undergo a socially regulated, highly stable lipid loss as part of their behavioral maturation. We used large-scale transcriptomic and proteomic experiments, physiological experiments and RNA interference to explore the mechanistic basis for this lipid loss. Lipid loss was associated with thousands of gene expression changes in abdominal fat bodies. Many of these genes were also regulated in young bees by nutrition during an initial period of lipid gain. Surprisingly, in older bees, which is when maximum lipid loss occurs, diet played less of a role in regulating fat body gene expression for components of evolutionarily conserved nutrition-related endocrine systems involving insulin and juvenile hormone signaling. By contrast, fat body gene expression in older bees was regulated more strongly by evolutionarily novel regulatory factors, queen mandibular pheromone (a honey bee-specific social signal) and vitellogenin (a conserved yolk protein that has evolved novel, maturation-related functions in the bee), independent of nutrition. These results demonstrate that conserved molecular pathways can be manipulated to achieve stable lipid loss through evolutionarily novel regulatory processes. PMID:22031746

Mechanisms of stable lipid loss in a social insect.

PubMed

Ament, Seth A; Chan, Queenie W; Wheeler, Marsha M; Nixon, Scott E; Johnson, S Peir; Rodriguez-Zas, Sandra L; Foster, Leonard J; Robinson, Gene E

2011-11-15

Worker honey bees undergo a socially regulated, highly stable lipid loss as part of their behavioral maturation. We used large-scale transcriptomic and proteomic experiments, physiological experiments and RNA interference to explore the mechanistic basis for this lipid loss. Lipid loss was associated with thousands of gene expression changes in abdominal fat bodies. Many of these genes were also regulated in young bees by nutrition during an initial period of lipid gain. Surprisingly, in older bees, which is when maximum lipid loss occurs, diet played less of a role in regulating fat body gene expression for components of evolutionarily conserved nutrition-related endocrine systems involving insulin and juvenile hormone signaling. By contrast, fat body gene expression in older bees was regulated more strongly by evolutionarily novel regulatory factors, queen mandibular pheromone (a honey bee-specific social signal) and vitellogenin (a conserved yolk protein that has evolved novel, maturation-related functions in the bee), independent of nutrition. These results demonstrate that conserved molecular pathways can be manipulated to achieve stable lipid loss through evolutionarily novel regulatory processes.

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

PubMed

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

2014-12-20

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

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

PubMed

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

2014-09-30

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

CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

PubMed Central

Poeschla, Eric M.; Looney, David J.

1998-01-01

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors. PMID:9658135

Model system for plant cell biology: GFP imaging in living onion epidermal cells

NASA Technical Reports Server (NTRS)

Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

1999-01-01

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

Different coding strategies for the perception of stable and changeable facial attributes.

PubMed

Taubert, Jessica; Alais, David; Burr, David

2016-09-01

Perceptual systems face competing requirements: improving signal-to-noise ratios of noisy images, by integration; and maximising sensitivity to change, by differentiation. Both processes occur in human vision, under different circumstances: they have been termed priming, or serial dependencies, leading to positive sequential effects; and adaptation or habituation, which leads to negative sequential effects. We reasoned that for stable attributes, such as the identity and gender of faces, the system should integrate: while for changeable attributes like facial expression, it should also engage contrast mechanisms to maximise sensitivity to change. Subjects viewed a sequence of images varying simultaneously in gender and expression, and scored each as male or female, and happy or sad. We found strong and consistent positive serial dependencies for gender, and negative dependency for expression, showing that both processes can operate at the same time, on the same stimuli, depending on the attribute being judged. The results point to highly sophisticated mechanisms for optimizing use of past information, either by integration or differentiation, depending on the permanence of that attribute.

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

PubMed

Casales, Erkuden; Aranda, Alejandro; Quetglas, Jose I; Ruiz-Guillen, Marta; Rodriguez-Madoz, Juan R; Prieto, Jesus; Smerdou, Cristian

2010-05-31

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells. 2010 Elsevier B.V. All rights reserved.

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

PubMed

Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

2015-01-01

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

Stable isotope deltas: Tiny, yet robust signatures in nature

USGS Publications Warehouse

Brand, Willi A.; Coplen, Tyler B.

2012-01-01

Although most of them are relatively small, stable isotope deltas of naturally occurring substances are robust and enable workers in anthropology, atmospheric sciences, biology, chemistry, environmental sciences, food and drug authentication, forensic science, geochemistry, geology, oceanography, and paleoclimatology to study a variety of topics. Two fundamental processes explain the stable isotope deltas measured in most terrestrial systems: isotopic fractionation and isotope mixing. Isotopic fractionation is the result of equilibrium or kinetic physicochemical processes that fractionate isotopes because of small differences in physical or chemical properties of molecular species having different isotopes. It is shown that the mixing of radioactive and stable isotope end members can be modelled to provide information on many natural processes, including 14C abundances in the modern atmosphere and the stable hydrogen and oxygen isotopic compositions of the oceans during glacial and interglacial times. The calculation of mixing fractions using isotope balance equations with isotope deltas can be substantially in error when substances with high concentrations of heavy isotopes (e.g. 13C, 2H, and 18O ) are mixed. In such cases, calculations using mole fractions are preferred as they produce accurate mixing fractions. Isotope deltas are dimensionless quantities. In the International System of Units (SI), these quantities have the unit 1 and the usual list of prefixes is not applicable. To overcome traditional limitations with expressing orders of magnitude differences in isotope deltas, we propose the term urey (symbol Ur), after Harold C. Urey, for the unit 1. In such a manner, an isotope delta value expressed traditionally as−25 per mil can be written as−25 mUr (or−2.5 cUr or−0.25 dUr; the use of any SI prefix is possible). Likewise, very small isotopic differences often expressed in per meg ‘units’ are easily included (e.g. either+0.015 ‰ or+15 per meg can be written as+15 μUr.

CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line.

PubMed

Yang, Maozhou; Zhang, Liang; Stevens, Jeff; Gibson, Gary

2014-12-01

The Swarm rat chondrosarcoma (RCS) cell lines derived from a spontaneous neoplasm in a rat spine several decades ago have provided excellent models of chondrosarcoma tumor development. In addition the robust chondrocyte phenotype (expression of a large panel of genes identical to that seen in normal rat cartilage) and the ability to generate cell clones have facilitated their extensive use in the identification of chondrocyte proteins and genes. The clustered regularly interspersed short palindromic repeat (CRISPR) technology employing the RNA-guided nuclease Cas9 has rapidly dominated the genome engineering field as a unique and powerful gene editing tool. We have generated a stable RCS cell line (RCS Cas9) expressing the nuclease Cas9 that enables the editing of any target gene or non-coding RNA by simple transfection with a guide RNA. As proof of principle, stable cell lines with targeted ablation of aggrecan expression (Acan KO) were generated and characterized. The studies show that stable chondrocyte cell lines with targeted genome editing can be quickly generated from RCS Cas9 cells using this system. The Acan KO cell lines also provided a tool for characterizing the response of chondrocytes to aggrecan loss and the role of aggrecan in chondrosarcoma development. Loss of aggrecan expression while not affecting the chondrocyte phenotype resulted in a much firmer attachment of cells to their substrate in culture. Large changes in the expression of several genes were observed in response to the absence of the proteoglycan matrix, including those for several small leucine rich proteoglycans (SLRPs), transcription factors and membrane transporters. Acan KO cells failed to form a substantial chondrosarcoma when injected subcutaneously in nude mice consistent with previous suggestions that the glycosaminoglycan-rich matrix surrounding the chondrosarcoma protects it from destruction by the host immune system. The studies provide new understanding of aggrecan function and the RCS Cas9 cell line is expected to provide a very valuable tool for the study gene function in chondrocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Stable Expression of the Motor Protein Prestin in Chinese Hamster Ovary Cells

NASA Astrophysics Data System (ADS)

Iida, Koji; Konno, Kazuaki; Oshima, Takeshi; Tsumoto, Kouhei; Ikeda, Katsuhisa; Kumagai, Izumi; Kobayashi, Toshimitsu; Wada, Hiroshi

Mammalian hearing sensitivity relies on a mechanical amplification mechanism involving the outer hair cells (OHCs), which rapidly alter their longitudinal length in response to changes in their membrane potential. The molecular basis of this mechanism is thought to be a motor protein embedded in the lateral membrane of the OHCs. Recently, this motor protein was identified and termed prestin. Since then, prestin has been researched intensively to elucidate the behavior of the OHCs. However, little progress in the study of prestin at the molecular level has been made because no method of obtaining an adequate amount of prestin has been established. In this study, therefore, an attempt was made to construct a stable expression system of prestin using Chinese hamster ovary (CHO) cells. The expression of prestin in the transfected CHO cells and the activity of prestin on CHO cells were confirmed by immunofluorescence and whole-cell patch-clamp measurements, respectively.

Establishment of stable cell line for inducing KAP1 protein expression.

PubMed

Liu, Xiaoyan; Khan, Md Asaduzzaman; Cheng, Jingliang; Wei, Chunli; Zhang, Lianmei; Fu, Junjiang

2015-06-01

Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by rDNA-mediated integration.

PubMed

Fang, Cheng; Wang, Qinhong; Selvaraj, Jonathan Nimal; Zhou, Yuling; Ma, Lixin; Zhang, Guimin; Ma, Yanhe

2017-08-18

Xylanase is a widely-used additive in baking industry for enhancing dough and bread quality. Several xylanases used in baking industry were expressed in different systems, but their expression in antibiotic free vector system is highly essential and safe. In the present study, an alternative rDNA-mediated technology was developed to increase the copy number of target gene by integrating it into Saccharomyces cerevisiae genome. A xylanase-encoding gene xynHB from Bacillus sp. was cloned into pHBM367H and integrated into S. cerevisiae genome through rDNA-mediated recombination. Exogenous XynHB expressed by recombinant S. cerevisiae strain A13 exhibited higher degradation activity towards xylan than other transformants. The real-time PCR analysis on A13 genome revealed the presence of 13.64 copies of xynHB gene. Though no antibiotics have been used, the genetic stability and the xylanase activity of xynHB remained stable up to 1,011 generations of cultivation. S. cerevisiae strain A13 expressing xylanase reduced the required kneading time and increased the height and diameter of the dough size, which would be safe and effective in baking industry as no antibiotics-resistance risk. The new effective rDNA-mediated technology without using antibiotics here provides a way to clone other food related industrial enzymes for applications.

pLR: a lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters.

PubMed

Vargas, José Eduardo; Salton, Gabrielle; Sodré de Castro Laino, Andressa; Pires, Tiago Dalberto; Bonamino, Martin; Lenz, Guido; Delgado-Cañedo, Andrés

2012-11-01

Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site)-- reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression. Copyright © 2012 Elsevier Inc. All rights reserved.

Uncarboxylated Osteocalcin and Gprc6a Axis Produce Intratumoral Androgens in Castration-Resistant Prostate Cancer

DTIC Science & Technology

2015-03-01

interacts with bone extracellular matrix associated calcium and hydroxyapatite and deposited in the bone matrix. Some Osteocalcin is released into...fluorescence protein as control) Osteocalcin and mutant Osteocalcin using lentivirus mediated stable infections. 2. Determined the gene expression of Gprc61... used a lentiviral system for expressing Osteocalcin and mutated Osteocalcin. Osteocalcin is mutated at three positions where glutamic acid residue at

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

PubMed

Chung, Nancy P Y; Matthews, Katie; Kim, Helen J; Ketas, Thomas J; Golabek, Michael; de Los Reyes, Kevin; Korzun, Jacob; Yasmeen, Anila; Sanders, Rogier W; Klasse, Per Johan; Wilson, Ian A; Ward, Andrew B; Marozsan, Andre J; Moore, John P; Cupo, Albert

2014-04-25

Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

Generating mammalian stable cell lines by electroporation.

PubMed

A Longo, Patti; Kavran, Jennifer M; Kim, Min-Sung; Leahy, Daniel J

2013-01-01

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr-Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method. Copyright © 2013 Elsevier Inc. All rights reserved.

Doxycycline-Regulated 3T3-L1 Preadipocyte Cell Line with Inducible, Stable Expression of Adenoviral E4orf1 Gene: A Cell Model to Study Insulin-Independent Glucose Disposal

PubMed Central

Krishnapuram, Rashmi; Dhurandhar, Emily J.; Dubuisson, Olga; Hegde, Vijay; Dhurandhar, Nikhil V.

2013-01-01

Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin. PMID:23544159

Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

PubMed

Krishnapuram, Rashmi; Dhurandhar, Emily J; Dubuisson, Olga; Hegde, Vijay; Dhurandhar, Nikhil V

2013-01-01

Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin.

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.

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

PubMed Central

Sinha, Pallavi; Singh, Vikas K.; Suryanarayana, V.; Krishnamurthy, L.; Saxena, Rachit K.; Varshney, Rajeev K.

2015-01-01

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions. PMID:25849964

Evaluation and validation of housekeeping genes as reference for gene expression studies in pigeonpea (Cajanus cajan) under drought stress conditions.

PubMed

Sinha, Pallavi; Singh, Vikas K; Suryanarayana, V; Krishnamurthy, L; Saxena, Rachit K; Varshney, Rajeev K

2015-01-01

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.

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

PubMed

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

2011-11-17

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

Advances in recombinant protein expression for use in pharmaceutical research.

PubMed

Assenberg, Rene; Wan, Paul T; Geisse, Sabine; Mayr, Lorenz M

2013-06-01

Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells. Copyright © 2013. Published by Elsevier Ltd.

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.

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

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

PubMed

Kido, Takumi; Horigome, Tomoatsu; Uda, Minori; Adachi, Naoki; Hirai, Yohei

2017-09-01

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit.

PubMed

Ruf, S; Hermann, M; Berger, I J; Carrer, H; Bock, R

2001-09-01

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.

Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

PubMed Central

2011-01-01

Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi-cassette vectors in combination with various inserts of interest. PMID:21435248

Characterization of electrical noise limits in ultra-stable laser systems.

PubMed

Zhang, J; Shi, X H; Zeng, X Y; Lü, X L; Deng, K; Lu, Z H

2016-12-01

We demonstrate thermal noise limited and shot noise limited performance of ultra-stable diode laser systems. The measured heterodyne beat linewidth between such two independent diode lasers reaches 0.74 Hz. The frequency instability of one single laser approaches 1.0 × 10 -15 for averaging time between 0.3 s and 10 s, which is close to the thermal noise limit of the reference cavity. Taking advantage of these two ultra-stable laser systems, we systematically investigate the ultimate electrical noise contributions, and derive expressions for the closed-loop spectral density of laser frequency noise. The measured power spectral density of the beat frequency is compared with the theoretically calculated closed-loop spectral density of the laser frequency noise, and they agree very well. It illustrates the power and generality of the derived closed-loop spectral density formula of the laser frequency noise. Our result demonstrates that a 10 -17 level locking in a wide frequency range is feasible with careful design.

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

PubMed

Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

2016-05-01

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intrinsic gene expression subsets of diffuse cutaneous systemic sclerosis are stable in serial skin biopsies

PubMed Central

Pendergrass, Sarah A.; Lemaire, Raphael; Francis, Ian; Mahoney, J. Matthew; Lafyatis, Robert; Whitfield, Michael L.

2012-01-01

Skin biopsy gene expression was analyzed by DNA microarray from 13 dSSc patients enrolled in an open label study of rituximab, 9 dSSc patients not treated with rituximab, and 9 healthy controls. These data recapitulate the patient ‘intrinsic’ gene expression subsets described previously including proliferation, inflammatory, and normal-like groups. Serial skin biopsies showed consistent and non-progressing gene expression over time, and importantly, the patients in the inflammatory subset do not move to the fibroproliferative subset, and vice versa. We were unable to detect significant differences in gene expression before and after rituximab treatment, consistent with an apparent lack of clinical response. Serial biopsies from each patient stayed within the same gene expression subset regardless of treatment regimen or the time point at which they were taken. Collectively, these data emphasize the heterogeneous nature of SSc and demonstrate that the intrinsic subsets are an inherent, reproducible and stable feature of the disease that is independent of disease duration. Moreover, these data have fundamental importance for the future development of personalized therapy for SSc; drugs targeting inflammation are likely to benefit those patients with an inflammatory signature, whereas drugs targeting fibrosis are likely to benefit those with a fibroproliferative signature. PMID:22318389

Selection and validation of reliable housekeeping genes to evaluate Piscirickettsia salmonis gene expression.

PubMed

Flores-Herrera, Patricio; Arredondo-Zelada, Oscar; Marshall, Sergio H; Gómez, Fernando A

2018-06-01

Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis. Copyright © 2018. Published by Elsevier B.V.

Guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results

USGS Publications Warehouse

Coplen, Tyler B.

2011-01-01

To minimize confusion in the expression of measurement results of stable isotope and gas-ratio measurements, recommendations based on publications of the Commission on Isotopic Abundances and Atomic Weights of the International Union of Pure and Applied Chemistry (IUPAC) are presented. Whenever feasible, entries are consistent with the Système International d'Unités, the SI (known in English as the International System of Units), and the third edition of the International Vocabulary of Basic and General Terms in Metrology (VIM, 3rd edition). The recommendations presented herein are approved by the Commission on Isotopic Abundances and Atomic Weights and are designed to clarify expression of quantities related to measurement of isotope and gas ratios to ensure that quantity equations instead of numerical value equations are used for quantity definitions. Examples of column headings consistent with quantity calculus (also called the algebra of quantities) and examples of various deprecated usages connected with the terms recommended are presented.

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

PubMed Central

2009-01-01

Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine. PMID:19930574

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.

PubMed

Lombardi, Raffaele; Circelli, Patrizia; Villani, Maria Elena; Buriani, Giampaolo; Nardi, Luca; Coppola, Valentina; Bianco, Linda; Benvenuto, Eugenio; Donini, Marcello; Marusic, Carla

2009-11-20

In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.

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

SABRE: a method for assessing the stability of gene modules in complex tissues and subject populations.

PubMed

Shannon, Casey P; Chen, Virginia; Takhar, Mandeep; Hollander, Zsuzsanna; Balshaw, Robert; McManus, Bruce M; Tebbutt, Scott J; Sin, Don D; Ng, Raymond T

2016-11-14

Gene network inference (GNI) algorithms can be used to identify sets of coordinately expressed genes, termed network modules from whole transcriptome gene expression data. The identification of such modules has become a popular approach to systems biology, with important applications in translational research. Although diverse computational and statistical approaches have been devised to identify such modules, their performance behavior is still not fully understood, particularly in complex human tissues. Given human heterogeneity, one important question is how the outputs of these computational methods are sensitive to the input sample set, or stability. A related question is how this sensitivity depends on the size of the sample set. We describe here the SABRE (Similarity Across Bootstrap RE-sampling) procedure for assessing the stability of gene network modules using a re-sampling strategy, introduce a novel criterion for identifying stable modules, and demonstrate the utility of this approach in a clinically-relevant cohort, using two different gene network module discovery algorithms. The stability of modules increased as sample size increased and stable modules were more likely to be replicated in larger sets of samples. Random modules derived from permutated gene expression data were consistently unstable, as assessed by SABRE, and provide a useful baseline value for our proposed stability criterion. Gene module sets identified by different algorithms varied with respect to their stability, as assessed by SABRE. Finally, stable modules were more readily annotated in various curated gene set databases. The SABRE procedure and proposed stability criterion may provide guidance when designing systems biology studies in complex human disease and tissues.

Assessment of long-term transgene expression in barley: Ds-mediated delivery of bar results in robust, stable, and heritable expression

USDA-ARS?s Scientific Manuscript database

The utility of transgenic plants for both experimental and practical agronomic purposes is highly dependent on stable, predictable, and heritable expression of the introduced genes. This requirement is frequently unfulfilled, and transgenes are frequently subject to silencing. Studies of the charact...

Metazoan tRNA introns generate stable circular RNAs in vivo

PubMed Central

Lu, Zhipeng; Filonov, Grigory S.; Noto, John J.; Schmidt, Casey A.; Hatkevich, Talia L.; Wen, Ying; Jaffrey, Samie R.; Matera, A. Gregory

2015-01-01

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating “designer” circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. PMID:26194134

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.

Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells.

PubMed

Chun, B H; Bang, W G; Park, Y K; Woo, S K

2001-11-01

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed.

An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives.

PubMed

Randolph, Lauren N; Bao, Xiaoping; Zhou, Chikai; Lian, Xiaojun

2017-05-08

Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies due to their unique combination of two properties: pluripotency and unlimited proliferative capacity. However, directed differentiation of hPSCs to clinically relevant cell lineages is needed to achieve the goal of hPSC-based therapies. This requires a deep understanding of how cell signaling pathways converge on the nucleus to control differentiation and the ability to dissect gene function in a temporal manner. Here, we report the use of the PiggyBac transposon and a Tet-On 3G drug-inducible gene expression system to achieve versatile inducible gene expression in hPSC lines. Our new system, XLone, offers improvement over previous Tet-On systems with significantly reduced background expression and increased sensitivity to doxycycline. Transgene expression in hPSCs is tightly regulated in response to doxycycline treatment. In addition, the PiggyBac elements in our XLone construct provide a rapid and efficient strategy for generating stable transgenic hPSCs. Our inducible gene expression PiggyBac transposon system should facilitate the study of gene function and directed differentiation in human stem cells.

Sunflower (Helianthus annuus L.).

PubMed

Radonic, Laura M; Lewi, Dalia M; López, Nilda E; Hopp, H Esteban; Escandón, Alejandro S; Bilbao, Marisa López

2015-01-01

Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and transformation in spite of the publication of different protocols. Here we describe a routine transformation system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105 strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important genes and also for the evaluation of the specific expression patterns of different promoters in transgenic sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation in several independent lines.

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

PubMed

Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik

2004-11-01

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

PubMed Central

Dai, Qun; Aleksandrov, Andrei A.; Bajrami, Bekim; Diego, Pamela Ann; Wu, Xing; Ray, Marjorie; Naren, Anjaparavanda P.; Riordan, John R.; Yao, Xudong; DeLucas, Lawrence J.; Urbatsch, Ina L.; Kappes, John C.

2015-01-01

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, or an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTRFLAG-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTRFLAG-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment futrure CF drug discovery efforts, including biophysical and structural studies. PMID:25577540

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

PubMed

Aswal, Ajay Pal Singh; Raghav, Sarvesh; De, Sachinandan; Thakur, Manish; Goswami, Surender Lal; Datta, Tirtha Kumar

2008-01-15

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

Cereal transformation through particle bombardment

NASA Technical Reports Server (NTRS)

Casas, A. M.; Kononowicz, A. K.; Bressan, R. A.; Hasegawa, P. M.; Mitchell, C. A. (Principal Investigator)

1995-01-01

The review focuses on experiments that lead to stable transformation in cereals using microprojectile bombardment. The discussion of biological factors that affect transformation examines target tissues and vector systems for gene transfer. The vector systems include reporter genes, selectable markers, genes of agronomic interest, and vector constructions. Other topics include physical parameters that affect DNA delivery, selection of stably transformed cells and plant regeneration, and analysis of gene expression and transmission to the progeny.

[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

PubMed

Wang, Wan; Zhang, Qing; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Zhang, Rongbo; Wu, Jing; Hu, Dong

2015-09-01

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

PubMed

Shirzad, Hedayatollah; Esmaeili, Fariba; Bakhshalizadeh, Shabnam; Ebrahimie, Marzieh; Ebrahimie, Esmaeil

2017-04-01

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP + ). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP + into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP + cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Metazoan tRNA introns generate stable circular RNAs in vivo.

PubMed

Lu, Zhipeng; Filonov, Grigory S; Noto, John J; Schmidt, Casey A; Hatkevich, Talia L; Wen, Ying; Jaffrey, Samie R; Matera, A Gregory

2015-09-01

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. © 2015 Lu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Brain selective transgene expression in zebrafish using an NRSE derived motif

PubMed Central

Bergeron, Sadie A.; Hannan, Markus C.; Codore, Hiba; Fero, Kandice; Li, Grace H.; Moak, Zachary; Yokogawa, Tohei; Burgess, Harold A.

2012-01-01

Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae. PMID:23293587

Is Approximate Number Precision a Stable Predictor of Math Ability?

ERIC Educational Resources Information Center

Libertus, Melissa E.; Feigenson, Lisa; Halberda, Justin

2013-01-01

Previous research shows that children's ability to estimate numbers of items using their Approximate Number System (ANS) predicts later math ability. To more closely examine the predictive role of early ANS acuity on later abilities, we assessed the ANS acuity, math ability, and expressive vocabulary of preschoolers twice, six months apart. We…

Elucidating differences in gene expression of Epichloe coenophiala endophytic fungus in reproductive vs. vegetative tissues of tall fescue (Lolium arundinaceum)

USDA-ARS?s Scientific Manuscript database

The tall fescue – Epichloe coenophiala symbiotic system is the most extensively studied of any grass-microbe symbiosis, mainly because of its economic importance worldwide. This is also an extraordinarily stable and mutualistic symbiosis where the endophyte colonizes both vegetative and reproductive...

Linking low-level stable isotope fractionation to expression of the cytochrome P450 monooxygenase-encoding ethB gene for elucidation of methyl tert-butyl ether biodegradation in aerated treatment pond systems.

PubMed

Jechalke, Sven; Rosell, Mònica; Martínez-Lavanchy, Paula M; Pérez-Leiva, Paola; Rohwerder, Thore; Vogt, Carsten; Richnow, Hans H

2011-02-01

Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [ε(C)] of -0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [ε(H)]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon (ε(C) of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (ε(H) of -5‰ ± 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem.

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

PubMed Central

Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

2016-01-01

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

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

PubMed

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

2017-01-01

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

Use of the Uteroglobin Platform for the Expression of a Bivalent Antibody against Oncofetal Fibronectin in Escherichia coli

PubMed Central

Ventura, Elisa; Riondato, Mattia; Sambuceti, Gianmario; Salis, Annalisa; Damonte, Gianluca; Cordazzo, Cinzia; Besir, Hüseyin; Pistoia, Vito; Zardi, Luciano

2013-01-01

Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646–26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50–70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli. PMID:24367567

[Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

PubMed

Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

2015-12-01

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems.

PubMed

Tremblay, Reynald; Feng, Mary; Menassa, Rima; Huner, Norman P A; Jevnikar, Anthony M; Ma, Shengwu

2011-04-01

Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs.

Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays.

PubMed

Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi

2017-01-01

Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T 1 and T 2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T 2 progenies and PCR-identified Apr gene segregation in T 2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods.

In Vivo Stable Transduction of Humanized Liver Tissue in Chimeric Mice via High-Capacity Adenovirus–Lentivirus Hybrid Vector

PubMed Central

Kataoka, Miho; Tateno, Chise; Yoshizato, Katsutoshi; Kawasaki, Yoshiko; Kimura, Takahiro; Faure-Kumar, Emmanuelle; Palmer, Donna J.; Ng, Philip; Okamura, Haruki; Kasahara, Noriyuki

2010-01-01

Abstract We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved stable transduction by the second-stage lentivirus produced in situ from first-stage adenovirus. This represents the first demonstration of the functionality of adenoviral-lentiviral hybrid vectors in a normal parenchymal organ in vivo. PMID:19725756

The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.

PubMed

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5'-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.

The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

PubMed Central

Sharma, Nynne; Hollensen, Anne Kruse; Bak, Rasmus O.; Staunstrup, Nicklas Heine; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2012-01-01

DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells. PMID:23110238

Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function.

PubMed

Trojan, Karina; Unterrainer, Christian; Weimer, Rolf; Bulut, Nuray; Morath, Christian; Aly, Mostafa; Zhu, Li; Opelz, Gerhard; Daniel, Volker

2017-01-01

There is circumstantial evidence that IFNy+ Treg might have clinical relevance in transplantation. IFNy+ Treg express IFNy receptors and are induced by IFNy. In the present study we investigated in kidney transplant recipients with good long-term stable graft function the absolute cell counts of IFNy+ Treg subsets and whether their expression of Foxp3 is stable or transient. Helios expression determined by eight-color-fluorescence flow cytometry and methylation status of the Foxp3 Treg specific demethylation region (TSDR) served as indicators for stability of Foxp3 expression. Methylation status was investigated in enriched IFNy+ and IFNy- Treg preparations originating from peripheral blood using high resolution melt analysis. A total of 136 transplant recipients and 52 healthy controls were studied. Proportions of IFNy+ Treg were similar in patients and healthy controls (0.05% and 0.04% of all CD4+ lymphocytes; p = n.s.). Patients also had similar absolute counts of IFNy producing Helios+ and Helios- Treg (p = n.s.). Most of the IFNy+ and IFNy- Treg in transplant recipients had a methylated Foxp3 TSDR, however, there was a sizeable proportion of IFNy+ and IFNy- Treg with demethylated Foxp3 TSDR. Male and female patients showed more frequently methylated IFNy+ and IFNy- Treg than male and female controls (all p<0.05). Kidney transplant recipients with good long-term stable graft function have similar levels of IFNy+ Treg as healthy controls. IFNy+ and IFNy- Treg subsets in patients consist of cells with stable and cells with transient Foxp3 expression; however, patients showed more frequently methylated IFNy+ and IFNy- Treg than controls. The data show increased levels of Treg subsets with stable as well as transient Foxp3 expression in patients with stable allograft acceptance compared to healthy controls.

The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression.

PubMed

Gasanov, N B; Toshchakov, S V; Georgiev, P G; Maksimenko, O G

2015-01-01

Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta

PubMed Central

de Jong, Danielle M.; Seaver, Elaine C.

2016-01-01

Regeneration, the ability to replace lost tissues and body parts following traumatic injury, occurs widely throughout the animal tree of life. Regeneration occurs either by remodeling of pre-existing tissues, through addition of new cells by cell division, or a combination of both. We describe a staging system for posterior regeneration in the annelid, Capitella teleta, and use the C. teleta Hox gene code as markers of regional identity for regenerating tissue along the anterior-posterior axis. Following amputation of different posterior regions of the animal, a blastema forms and by two days, proliferating cells are detected by EdU incorporation, demonstrating that epimorphosis occurs during posterior regeneration of C. teleta. Neurites rapidly extend into the blastema, and gradually become organized into discrete nerves before new ganglia appear approximately seven days after amputation. In situ hybridization shows that seven of the ten Hox genes examined are expressed in the blastema, suggesting roles in patterning the newly forming tissue, although neither spatial nor temporal co-linearity was detected. We hypothesized that following amputation, Hox gene expression in pre-existing segments would be re-organized to scale, and the remaining fragment would express the complete suite of Hox genes. Surprisingly, most Hox genes display stable expression patterns in the ganglia of pre-existing tissue following amputation at multiple axial positions, indicating general stability of segmental identity. However, the three Hox genes, CapI-lox4, CapI-lox2 and CapI-Post2, each shift its anterior expression boundary by one segment, and each shift includes a subset of cells in the ganglia. This expression shift depends upon the axial position of the amputation. In C. teleta, thoracic segments exhibit stable positional identity with limited morphallaxis, in contrast with the extensive body remodeling that occurs during regeneration of some other annelids, planarians and acoel flatworms. PMID:26894631

Insular Activity during Passive Viewing of Aversive Stimuli Reflects Individual Differences in State Negative Affect

ERIC Educational Resources Information Center

Meriau, Katja; Wartenburger, Isabell; Kazzer, Philipp; Prehn, Kristin; Villringer, Arno; van der Meer, Elke; Heekeren, Hauke R.

2009-01-01

People differ with regard to how they perceive, experience, and express negative affect. While trait negative affect reflects a stable, sustained personality trait, state negative affect represents a stimulus limited and temporally acute emotion. So far, little is known about the neural systems mediating the relationship between negative affect…

Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein

PubMed Central

Fiedler, Nicola; Quant, Ellen; Fink, Ludger; Sun, Jianguang; Schuster, Ralph; Gerlich, Wolfram H; Schaefer, Stephan

2006-01-01

AIM: Hepatitis B virus protein X (HBx) has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type. METHODS: We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation. RESULTS: The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21waf/cip/sdi which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2. In AML-HBx9 cells, p21waf/cip/sdi-protein expression and p21waf/cip/sdi transcription were deregulated. Furthermore, the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation. Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines. CONCLUSION: Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis. PMID:16937438

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor.

PubMed

Hassaïne, Ghérici; Deluz, Cédric; Grasso, Luigino; Wyss, Romain; Hovius, Ruud; Stahlberg, Henning; Tomizaki, Takashi; Desmyter, Aline; Moreau, Christophe; Peclinovska, Lucie; Minniberger, Sonja; Mebarki, Lamia; Li, Xiao-Dan; Vogel, Horst; Nury, Hugues

2017-01-01

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-01-01

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

Production of membrane proteins without cells or detergents.

PubMed

Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael

2011-04-30

The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2017-12-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-07-01

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

Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression

PubMed Central

Tonack, Sarah; Patel, Sabina; Jalali, Mehdi; Nedjadi, Taoufik; Jenkins, Rosalind E; Goldring, Christopher; Neoptolemos, John; Costello, Eithne

2011-01-01

AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised. RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable. PMID:21528072

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.

Modeling sRNA-Regulated Plasmid Maintenance

PubMed Central

Klumpp, Stefan

2017-01-01

We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin’s mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed. PMID:28085919

A stable RNA virus-based vector for citrus trees

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

Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe

Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter.more » These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.« less

Generation of a stable cell line for constitutive miRNA expression.

PubMed

Lieber, Diana

2013-01-01

miRNAs have in recent years emerged as novel players in virus-host interactions. While individual miRNAs are capable of regulating many targets simultaneously, not much is known about the role of distinct host or viral miRNAs in the context of infection. Analysis of the function of a miRNA is often hampered by the complexity of virus-host interactions and the enormous changes in the host cell during infection. Many viral miRNAs as for example from Kaposi sarcoma-associated Herpesvirus (KSHV) are probably exclusively expressed in latent infection. This might lead to a steady-state situation with offense and defense mechanisms counteracting each other. Cellular miRNAs involved in defense against pathogens on the other hand might be suppressed in infection. A cell culture system allowing for constitutive expression of individual miRNAs at high levels is a useful tool to enhance miRNA-specific functions and to uncouple viral miRNA function from other infection-related mechanisms. Here, a protocol is described to generate stable cell lines for constitutive expression of single cellular or viral miRNA precursors in absence of infection. The procedure comprises cloning of the precursor sequence, generation of the lentiviral expression vector, transduction of the cells of interest, selection for polyclonal cell lines, and isolation of monoclonal cell lines by limiting dilution.

Influence of initial seed distribution on the pattern formation of the phase field crystals

NASA Astrophysics Data System (ADS)

Starodumov, Ilya; Galenko, Peter; Kropotin, Nikolai; Alexandrov, Dmitri V.

2017-11-01

The process of crystal growth can be expressed as a transition of atomic structure to a finally stable state or to a metastable state. In the Phase Field Crystal Model (PFC-model) these states are described by regular distributions of the atomic density. Getting the system into any metastable condition may be caused by the peculiarities of the computational domain, initial and boundary conditions. However, an important factor in the formation of the crystal structure can be the initial disturbance. In the report we show how different types of initial disturbance can change the finally stable state of crystal structure in equilibrium.

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II.

PubMed Central

Handl, C; Rönnberg, B; Nilsson, B; Olsson, E; Jonsson, H; Flock, J I

1988-01-01

The gene for Escherichia coli heat-stable enterotoxin type II (STII) was fused to the genes for protein A from Staphylococcus aureus and beta-galactosidase in two different expression systems. Antibodies raised in rabbits against the protein A-STII fusion protein recognized the beta-galactosidase-STII fusion protein. The latter fusion protein was used as the immobilized antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of STII. The correlation between the results of the ELISA and the intestinal loop test in piglets was 95%, suggesting that the ELISA can be used to reliably detect STII. Images PMID:3049659

InSAR datum connection using GNSS-augmented radar transponders

NASA Astrophysics Data System (ADS)

Mahapatra, Pooja; der Marel, Hans van; van Leijen, Freek; Samiei-Esfahany, Sami; Klees, Roland; Hanssen, Ramon

2018-01-01

Deformation estimates from Interferometric Synthetic Aperture Radar (InSAR) are relative: they form a `free' network referred to an arbitrary datum, e.g. by assuming a reference point in the image to be stable. However, some applications require `absolute' InSAR estimates, i.e. expressed in a well-defined terrestrial reference frame, e.g. to compare InSAR results with those of other techniques. We propose a methodology based on collocated InSAR and Global Navigation Satellite System (GNSS) measurements, achieved by rigidly attaching phase-stable millimetre-precision compact active radar transponders to GNSS antennas. We demonstrate this concept through a simulated example and practical case studies in the Netherlands.

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

PubMed

Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

2016-01-29

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effects of angiotensin-converting enzyme inhibitor versus valsartan on cellular signaling events in heart transplant.

PubMed

White, Michel; Ross, Heather; Levesque, Sylvie; Whittom, Lucette; Pelletier, Guy B; Racine, Normand; Meloche, Sylvain; Voisin, Laure

2009-05-01

Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) provide similar biologic effects in model systems and similar clinical impacts in humans. The changes in the cardiac angiotensin system signaling pathways in the human heart in response to ACE inhibitors versus ARBs have been incompletely studied. To investigate the effects of ACE inhibitors versus valsartan on the angiotensin II signal transduction pathways in the transplanted human heart. Twenty-seven stable cardiac transplant recipients were randomized to remain on ACE inhibitor therapy (n = 8) or to receive valsartan (n = 19). Two additional endomyocardial biopsy samples were obtained at baseline and after 9 months of therapy. The expression of cardiac angiotensin type I and II receptors and atrial natriuretic factor (ANF) was measured by quantitative polymerase chain reaction. The expression and phosphorylation levels of selected signal transduction pathways were analyzed by immunoblotting. The mean dose of valsartan was 114 +/- 41 mg/day. The use of valsartan resulted in a similar impact on blood pressure and biochemistry profile. There were no significant changes in the expression of angiotensin type I and II receptors and ANF with valsartan. Similarly, no significant changes in the expression and phosphorylation of Jun N-terminal kinase, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinases or AKT, and mammalian target of rapamycin was observed in the valsartan-treated group. Valsartan use is associated with similar clinical and molecular cardiac effects as ACE inhibitor therapy in stable long-term cardiac transplant recipients.

Expression and crystallization of the plant alternative oxidase.

PubMed

May, Benjamin; Elliott, Catherine; Iwata, Momi; Young, Luke; Shearman, Julia; Albury, Mary S; Moore, Anthony L

2015-01-01

The alternative oxidase (AOX) is an integral monotopic membrane protein located on the inner surface of the inner mitochondrial membrane. Branching from the traditional respiratory chain at the quinone pool, AOX is responsible for cyanide-resistant respiration in plants and fungi, heat generation in thermogenic plants, and survival of parasites, such as Trypanosoma brucei, in the human host. A recently solved AOX structure provides insight into its active site, thereby facilitating rational phytopathogenic and antiparasitic drug design. Here, we describe expression of recombinant AOX using two different expression systems. Purification protocols for the production of highly pure and stable AOX protein in sufficient quantities to facilitate further kinetic, biophysical, and structural analyses are also described.

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

PubMed

He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

2015-10-01

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

External optical imaging of freely moving mice with green fluorescent protein-expressing metastatic tumors

NASA Astrophysics Data System (ADS)

Yang, Meng; Baranov, Eugene; Shimada, Hiroshi; Moossa, A. R.; Hoffman, Robert M.

2000-04-01

We report here a new approach to genetically engineering tumors to become fluorescence such that they can be imaged externally in freely-moving animals. We describe here external high-resolution real-time fluorescent optical imaging of metastatic tumors in live mice. Stable high-level green flourescent protein (GFP)-expressing human and rodent cell lines enable tumors and metastasis is formed from them to be externally imaged from freely-moving mice. Real-time tumor and metastatic growth were quantitated from whole-body real-time imaging in GFP-expressing melanoma and colon carcinoma models. This GFP optical imaging system is highly appropriate for high throughput in vivo drug screening.

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples

PubMed Central

Zubakov, Dmitry; Hanekamp, Eline; Kokshoorn, Mieke; van IJcken, Wilfred

2007-01-01

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice. Electronic supplementary material The online version of this article (doi:10.1007/s00414-007-0182-6) contains supplementary material, which is available to authorized users. PMID:17579879

Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

PubMed

Cunningham, Sharon C; Siew, Susan M; Hallwirth, Claus V; Bolitho, Christine; Sasaki, Natsuki; Garg, Gagan; Michael, Iacovos P; Hetherington, Nicola A; Carpenter, Kevin; de Alencastro, Gustavo; Nagy, Andras; Alexander, Ian E

2015-08-01

Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration. © 2015 by the American Association for the Study of Liver Diseases.

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate.

PubMed

Park, Soohyun; Hong, Soohye; Pack, Seung Pil; Lee, Jinwon

2014-02-01

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5-30 °C) and weakly basic conditions (pH 8.5-10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.

Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

Cancer.gov

The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic

Nonlinear Stimulated Raman Exact Passage by Resonance-Locked Inverse Engineering

NASA Astrophysics Data System (ADS)

Dorier, V.; Gevorgyan, M.; Ishkhanyan, A.; Leroy, C.; Jauslin, H. R.; Guérin, S.

2017-12-01

We derive an exact and robust stimulated Raman process for nonlinear quantum systems driven by pulsed external fields. The external fields are designed with closed-form expressions from the inverse engineering of a given efficient and stable dynamics. This technique allows one to induce a controlled population inversion which surpasses the usual nonlinear stimulated Raman adiabatic passage efficiency.

Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies

PubMed Central

Zhu, Jinge; Rao, Hongyu; Tonelli, Marco; Westler, Milo; Singarapu, Kiran K.; Markley, John L.; DeLuca, Hector F.; Assadi-Porter, Fariba M.

2012-01-01

Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from E. coli inclusion bodies. The heterologously expressed protein constructs retain full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with, 13C, and 15N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed. PMID:22750673

Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

PubMed

Kanamoto, Hirosuke; Yamashita, Atsushi; Asao, Hiroshi; Okumura, Satoru; Takase, Hisabumi; Hattori, Masahira; Yokota, Akiho; Tomizawa, Ken-Ichi

2006-04-01

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

[Detection of stable expression of human interlukin-2 gene in transfected keratinocytes].

PubMed

Liao, W; Liu, Y; Ye, L

1999-09-01

To investigate the stable expression and secretion of human interlukin-2 gene in transfected keratinocytes. Keratinocytes were transfected with lipofectamine and selected by G418. Then the samples were analyzed with the techniques of DNA dot blot, RNA dot blot, hybridization in situ, immunohistochemistry, Western blot and MTT. The positive signals were observed in transfected keratinocytes by DNA dot blot, RNA dot blot, hybridization in situ and immunohistochemistry. With Western blot analysis, a specific band exhibiting a molecular weight of 15,000 was detected in transfected keratinocytes, which was in acordance with that of IL-2. The expression of IL-2 can maintain for up to 1 month. The amounts of IL-2 in the supernatants of two and four passages transfected keratinocytes were 27.7 U/ml and 15.0 U/ml, respectively. Keratinocytes have the potential for stable gene expression and secretion of active transgene products. Thus, it is possible to use keratinocytes as a target cell for gene transfection, gene expression and even gene therapy.

Intact Rapid Facial Mimicry as well as Generally Reduced Mimic Responses in Stable Schizophrenia Patients

PubMed Central

Chechko, Natalya; Pagel, Alena; Otte, Ellen; Koch, Iring; Habel, Ute

2016-01-01

Spontaneous emotional expressions (rapid facial mimicry) perform both emotional and social functions. In the current study, we sought to test whether there were deficits in automatic mimic responses to emotional facial expressions in patients (15 of them) with stable schizophrenia compared to 15 controls. In a perception-action interference paradigm (the Simon task; first experiment), and in the context of a dual-task paradigm (second experiment), the task-relevant stimulus feature was the gender of a face, which, however, displayed a smiling or frowning expression (task-irrelevant stimulus feature). We measured the electromyographical activity in the corrugator supercilii and zygomaticus major muscle regions in response to either compatible or incompatible stimuli (i.e., when the required response did or did not correspond to the depicted facial expression). The compatibility effect based on interactions between the implicit processing of a task-irrelevant emotional facial expression and the conscious production of an emotional facial expression did not differ between the groups. In stable patients (in spite of a reduced mimic reaction), we observed an intact capacity to respond spontaneously to facial emotional stimuli. PMID:27303335

Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle.

PubMed

Zhu, Wuzheng; Lin, Yaqiu; Liao, Honghai; Wang, Yong

2015-01-01

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

Directed chromosomal integration and expression of porcine rotavirus outer capsid protein VP4 in Lactobacillus casei ATCC393.

PubMed

Yin, Ji-Yuan; Guo, Chao-Qun; Wang, Zi; Yu, Mei-Ling; Gao, Shuai; Bukhari, Syed M; Tang, Li-Jie; Xu, Yi-Gang; Li, Yi-Jing

2016-11-01

Using two-step plasmid integration in the presence of 5-fluorouracil (5-FU), we developed a stable and markerless Lactobacillus casei strain for vaccine antigen expression. The upp of L. casei, which encodes uracil phosphoribosyltransferase (UPRTase), was used as a counterselection marker. We employed the Δupp isogenic mutant, which is resistant to 5-FU, as host and a temperature-sensitive suicide plasmid bearing upp expression cassette as counterselectable integration vector. Extrachromosomal expression of UPRTase complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. The resultant genotype can either be wild type or recombinant. The efficacy of the system was demonstrated by insertion and expression of porcine rotavirus (PRV) VP4. To improve VP4 expression, we analyzed L. casei transcriptional profiles and selected the constitutive highly expressed enolase gene (eno). The VP4 inserted after the eno termination codon were screened in the presence of 5-FU. Using genomic PCR amplification, we confirmed that VP4 was successfully integrated and stably inherited for at least 50 generations. Western blot demonstrated that VP4 was steadily expressed in medium with different carbohydrates. RT-qPCR and ELISA analysis showed that VP4 expression from the chromosomal location was similar to that achieved by a plasmid expression system. Applying the recombinant strain to immunize BALB/c mice via oral administration revealed that the VP4-expressing L. casei could induce both specific local and systemic humoral immune responses in mice. Overall, the improved gene replacement system represents an efficient method for chromosome recombination in L. casei and provides a safe tool for vaccine production.

Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo.

PubMed

De Bari, Cosimo; Dell'Accio, Francesco; Luyten, Frank P

2004-01-01

We previously reported the identification in a nude mouse assay of molecular markers predictive of the capacity of articular cartilage-derived cells (ACDCs) to form ectopic stable cartilage that is resistant to vascular invasion and endochondral ossification. In the present study, we investigated whether in vitro-differentiated mesenchymal stem cells (MSCs) from the synovial membrane (SM) express the stable-chondrocyte markers and form ectopic stable cartilage in vivo. Chondrogenesis was induced in micromass culture with the addition of transforming growth factor beta1 (TGFbeta1). After acquisition of the cartilage phenotype, micromasses were implanted subcutaneously into nude mice. Alternatively, cells were released enzymatically and either replated in monolayer or injected intramuscularly into nude mice. Marker analysis was performed by quantitative reverse transcription-polymerase chain reaction. Cell death was detected with TUNEL assay. Cartilage-like micromasses and released cells expressed the stable-chondrocyte markers at levels comparable with those expressed by stable ACDCs. The released cells lost chondrocyte marker expression by 24 hours in monolayer and failed to form cartilage when injected intramuscularly into nude mice. Instead, myogenic differentiation was detected. When intact TGFbeta1-treated micromasses were implanted subcutaneously, they partially lost their cartilage phenotype and underwent cell death and neoangiogenesis within 1 week. At later time points (15-40 days), we retrieved neither cartilage nor bone, and human cells were not detectable. The chondrocyte-like phenotype of human SM MSCs, induced in vitro under specific conditions, appears to be unstable and is not sufficient to obtain ectopic formation of stable cartilage in vivo. Studies in animal models of joint surface defect repair are necessary to evaluate the stability of the SM MSC chondrocyte-like phenotype within the joint environment.

Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis).

PubMed

Ashish, Shende; Bhure, S K; Harikrishna, Pillai; Ramteke, S S; Muhammed Kutty, V H; Shruthi, N; Ravi Kumar, G V P P S; Manish, Mahawar; Ghosh, S K; Mihir, Sarkar

2017-04-01

The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines. Copyright © 2017 Elsevier Inc. All rights reserved.

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 high-throughput immobilized bead screen for stable proteins and multi-protein complexes

PubMed Central

Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2011-01-01

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

Uncertain pollination environment promotes the evolution of a stable mixed reproductive system in the self-incompatible Hypochaeris salzmanniana (Asteraceae).

PubMed

Arista, M; Berjano, R; Viruel, J; Ortiz, M Á; Talavera, M; Ortiz, P L

2017-09-01

The transition from outcrossing to selfing is a repeated pattern in angiosperm diversification and according to general theory this transition should occur quickly and mixed reproductive systems should be infrequent. However, a large proportion of flowering plants have mixed reproductive systems, even showing inbreeding depression. Recently, several theoretical studies have shown that mixed mating systems can be stable, but empirical studies supporting these assumptions are still scarce. Hypochaeris salzmanniana, an annual species with populations differing in their self-incompatibility expression, was used as a study case to assess the stability of its mixed reproductive system. Here a descriptive study of the pollination environment was combined with measurements of the stability of the self-incompatibility system, outcrossing rate, reproductive assurance and inbreeding depression in four populations for two consecutive years. The reproductive system of populations exhibited a geographical pattern: the proportion of plants decreased from west to east. Pollinator environment also varied geographically, being less favourable from west to east. The self-incompatibility expression of some populations changed markedly in only one year. After selfing, progeny was mainly self-compatible, while after outcrossing both self-incompatible and self-compatible plants were produced. In general, both reproductive assurance and high inbreeding depression were found in all populations and years. The lowest values of inbreeding depression were found in 2014 in the easternmost populations, which experienced a marked increase in self-compatibility in 2015. The mixed reproductive system of H. salzmanniana seems to be an evolutionarily stable strategy, with selfing conferring reproductive assurance when pollinator attendance is low, but strongly limited by inbreeding depression. The fact that the highest frequencies of self-compatible plants appeared in the environments most unfavourable to pollination suggests that these plants are selected in these sites, although high rates of inbreeding depression should impede the complete loss of self-incompatibility. In H. salzmanniana, year-to-year changes in the frequency of self-incompatible individuals are directly derived from the balance between reproductive assurance and inbreeding depression. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please email: journals.permissions@oup.com

The information capacity of hypercycles.

PubMed

Silvestre, Daniel A M M; Fontanari, José F

2008-10-21

Hypercycles are information integration systems which are thought to overcome the information crisis of prebiotic evolution by ensuring the coexistence of several short templates. For imperfect template replication, we derive a simple expression for the maximum number of distinct templates n(m) that can coexist in a hypercycle and show that it is a decreasing function of the length L of the templates. In the case of high replication accuracy we find that the product n(m)L tends to a constant value, limiting thus the information content of the hypercycle. Template coexistence is achieved either as a stationary equilibrium (stable fixed point) or a stable periodic orbit in which the total concentration of functional templates is nonzero. For the hypercycle system studied here we find numerical evidence that the existence of an unstable fixed point is a necessary condition for the presence of periodic orbits.

An assessment of non-stationarity in physiological cognitive state assessment using artificial neural networks.

PubMed

Estepp, Justin R; Klosterman, Samantha L; Christensen, James C

2011-01-01

With increased attention toward physiological cognitive state assessment as a component in the larger field of applied neuroscience, the need to develop methods for robust, stable assessment of cognitive state has been expressed as critical to designing effective augmented human-machine systems. The technique of cognitive state assessment, as well as its benefits, has been demonstrated by many research groups. In an effort to move closer toward a realized system, efforts must now be focused on critical issues that remain unsolved, namely instability of pattern classifiers over the course of hours and days. This work, as part of the Cognitive State Assessment Competition 2011, seeks to explore methods for 'learning' non-stationarity as a mitigation for more generalized patterns that are stable over time courses that are not widely discussed in the literature.

Supermolecular drug challenge to overcome drug resistance in cancer cells.

PubMed

Onishi, Yasuhiko; Eshita, Yuki; Ji, Rui-Cheng; Kobayashi, Takashi; Onishi, Masayasu; Mizuno, Masaaki; Yoshida, Jun; Kubota, Naoji

2018-06-04

Overcoming multidrug resistance (MDR) of cancer cells can be accomplished using drug delivery systems in large-molecular-weight ATP-binding cassette transporters before entry into phagolysosomes and by particle-cell-surface interactions. However, these hypotheses do not address the intratumoral heterogeneity in cancer. Anti-MDR must be related to alterations of drug targets, expression of detoxification, as well as altered proliferation. In this study, it is shown that the excellent efficacy and sustainability of anti-MDR is due to a stable ES complex because of the allosteric facilities of artificial enzymes when they are used as supramolecular complexes. The allosteric effect of supermolecular drugs can be explained by the induced-fit model and can provide stable feedback control systems through the loop transfer function of the Hill equation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Better Bet-Hedging with coupled positive and negative feedback loops

NASA Astrophysics Data System (ADS)

Narula, Jatin; Igoshin, Oleg

2011-03-01

Bacteria use the phenotypic heterogeneity associated with bistable switches to distribute the risk of activating stress response strategies like sporulation and persistence. However bistable switches offer little control over the timing of phenotype switching and first passage times (FPT) for individual cells are found to be exponentially distributed. We show that a genetic circuit consisting of interlinked positive and negative feedback loops allows cells to control the timing of phenotypic switching. Using a mathematical model we find that in this system a stable high expression state and stable low expression limit cycle coexist and the FPT distribution for stochastic transitions between them shows multiple peaks at regular intervals. A multimodal FPT distribution allows cells to detect the persistence of stress and control the rate of phenotype transition of the population. We further show that extracellular signals from cell-cell communication that change the strength of the feedback loops can modulate the FPT distribution and allow cells even greater control in a bet-hedging strategy.

A simple method to generate stable cell lines for the analysis of transient protein-protein interactions.

PubMed

Savage, Emilia Elizabeth; Wootten, Denise; Christopoulos, Arthur; Sexton, Patrick Michael; Furness, Sebastian George Barton

2013-04-01

Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energy transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins.

Fast-slow asymptotic for semi-analytical ignition criteria in FitzHugh-Nagumo system.

PubMed

Bezekci, B; Biktashev, V N

2017-09-01

We study the problem of initiation of excitation waves in the FitzHugh-Nagumo model. Our approach follows earlier works and is based on the idea of approximating the boundary between basins of attraction of propagating waves and of the resting state as the stable manifold of a critical solution. Here, we obtain analytical expressions for the essential ingredients of the theory by singular perturbation using two small parameters, the separation of time scales of the activator and inhibitor and the threshold in the activator's kinetics. This results in a closed analytical expression for the strength-duration curve.

Quality assurance after process changes of the production of a therapeutic antibody.

PubMed

Brass, J M; Krummen, K; Moll-Kaufmann, C

1996-12-01

Process development for the production of a therapeutic humanised antibody is a very complex operation. It involves recombinant genetics, verification of a strong expression system, gene amplification, characterisation of a stable host cell expression system, optimisation and design of the mammalian cell culture fermentation system and development of an efficient recovery process resulting in high yields and product quality. Rapid progress in the field and the wish of some pharmaceutical companies for outsourcing their production are the driving forces for process changes relatively late in the development phase. This literature survey is aimed at identifying the limits of acceptable process changes in up scaling of the fermentation and down stream processing of biopharmaceuticals and defining the demand in production validation to prove product equivalency and identity of the isolated, purified therapeutic antibody.

[Lentivirus-mediated shRNA silencing of LAMP2A inhibits the proliferation of multiple myeloma cells].

PubMed

Li, Lixuan; Li, Jia

2015-05-01

To study the effects of lentivirus-mediated short hairpin RNA (shRNA) silencing of lysosome-associated membrane protein type 2A (LAMP2A) expression on the proliferation of multiple myeloma cells. The constructed shRNA lentiviral vector was applied to infect human multiple myeloma cell line MM.1S, and stable expression cell line was obtained by puromycin screening. Western blotting was used to verify the inhibitory effect on LAMP2A protein expression. MTT assay was conducted to detect the effect of knocked-down LAMP2A on MM.1S cell proliferation, and the anti-tumor potency of suberoylanilide hydroxamic acid (SAHA) against the obtained MM.1S LAMP2A(shRNA) stable cell line. Lactate assay was performed to observe the impact of low LAMP2A expression on cell glycolysis. The stable cell line with low LAMP2A expression were obtained with the constructed human LAMP2A-shRNA lentiviral vector. Down-regulation of LAMP2A expression significantly inhibited MM.1S cell proliferation and enhanced the anti-tumor activity of SAHA. Interestingly, decreased LAMP2A expression also inhibited MM.1S cell lactic acid secretion. Down-regulation of LAMP2A expression could inhibit cell proliferation in multiple myeloma cells.

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.

PubMed

Wilke, Sonja; Krausze, Joern; Gossen, Manfred; Groebe, Lothar; Jäger, Volker; Gherardi, Ermanno; van den Heuvel, Joop; Büssow, Konrad

2010-06-01

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.

PubMed

Aranda, Alejandro; Bezunartea, Jaione; Casales, Erkuden; Rodriguez-Madoz, Juan R; Larrea, Esther; Prieto, Jesus; Smerdou, Cristian

2014-12-01

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective.

PubMed

Yang, Bo; Liu, Jin; Jiang, Yue; Chen, Feng

2016-10-01

The species of Chlorella represent a highly specialized group of green microalgae that can produce high levels of protein. Many Chlorella strains can grow rapidly and achieve high cell density under controlled conditions and are thus considered to be promising protein sources. Many advances in the genetic engineering of Chlorella have occurred in recent years, with significant developments in successful expression of heterologous proteins for various applications. Nevertheless, a lot of obstacles remain to be addressed, and a sophisticated and stable Chlorella expression system has yet to emerge. This review provides a brief summary of current knowledge on Chlorella and an overview of recent progress in the genetic engineering of Chlorella, and highlights the advances in the development of a genetic toolbox of Chlorella for heterologous protein expression. Research directions to further exploit the Chlorella expression system with respect to both challenges and perspectives are also discussed. This paper serves as a comprehensive literature review for the Chlorella community and will provide valuable insights into future exploration of Chlorella as a promising host for heterologous protein expression. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection.

PubMed

Dreesen, Imke A J; Charpin-El Hamri, Ghislaine; Fussenegger, Martin

2010-02-01

While 15 million deaths per year are caused by communicable pathogens worldwide, health care authorities emphasize the considerable impact of poverty on the incidence of infectious diseases. The emergence of antigen-expressing plant tissues (e.g. rice, tomato, potato) has indicated the potential of land plants for low-cost vaccines in oral immunization programs. In this study, we engineered the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii for the stable expression of the D2 fibronectin-binding domain of Staphylococcus aureus fused with the cholera toxin B subunit (CTB), under the control of rbcL UTRs. Analysis of sera and faeces of mice, fed for 5 weeks with transgenic algae grown in confined Wave Bioreactor, revealed the induction of specific mucosal and systemic immune responses. Algae-based vaccination significantly reduced the pathogen load in the spleen and the intestine of treated mice and protected 80% of them against lethal doses of S. aureus. Importantly, the alga vaccine was stable for more than 1.5 years at room temperature. These results indicate that C. reinhardtii may play an important role in molecular pharming, as it combines the beneficial features of land plant vaccines, while offering unmatched ease of growth compared to other members of the plant kingdom. Copyright 2010 Elsevier B.V. All rights reserved.

Upregulation of microRNA 142-3p in the peripheral blood and urinary cells of kidney transplant recipients with post-transplant graft dysfunction

PubMed Central

Domenico, T.D.; Joelsons, G.; Montenegro, R.M.; Manfro, R.C.

2017-01-01

We analyzed microRNA (miR)-142-3p expression in leucocytes of the peripheral blood and urinary sediment cell samples obtained from kidney transplant recipients who developed graft dysfunction. Forty-one kidney transplant recipients with kidney graft dysfunction and 8 stable patients were included in the study. The groups were divided according to histological analysis into acute rejection group (n=23), acute tubular necrosis group (n=18) and stable patients group used as a control for gene expression (n=8). Percutaneous biopsies were performed and peripheral blood samples and urine samples were obtained. miR-142-3p was analyzed by real-time polymerase chain reaction. The group of patients with acute tubular necrosis presented significantly higher expressions in peripheral blood (P<0.05) and urine (P<0.001) compared to the stable patients group. Also, in the peripheral blood, miR-142-3p expression was significantly higher in the acute tubular necrosis group compared to the acute rejection group (P<0.05). Urine samples of the acute rejection group presented higher expression compared to the stable patients group (P<0.001) but the difference between acute tubular necrosis and acute rejection groups was not significant in the urinary analyzes (P=0.079). miR-142-3p expression has a distinct pattern of expression in the setting of post-operative acute tubular necrosis after kidney transplantation and may potentially be used as a non-invasive biomarker for renal graft dysfunction. PMID:28380212

Upregulation of microRNA 142-3p in the peripheral blood and urinary cells of kidney transplant recipients with post-transplant graft dysfunction.

PubMed

Domenico, T D; Joelsons, G; Montenegro, R M; Manfro, R C

2017-04-03

We analyzed microRNA (miR)-142-3p expression in leucocytes of the peripheral blood and urinary sediment cell samples obtained from kidney transplant recipients who developed graft dysfunction. Forty-one kidney transplant recipients with kidney graft dysfunction and 8 stable patients were included in the study. The groups were divided according to histological analysis into acute rejection group (n=23), acute tubular necrosis group (n=18) and stable patients group used as a control for gene expression (n=8). Percutaneous biopsies were performed and peripheral blood samples and urine samples were obtained. miR-142-3p was analyzed by real-time polymerase chain reaction. The group of patients with acute tubular necrosis presented significantly higher expressions in peripheral blood (P<0.05) and urine (P<0.001) compared to the stable patients group. Also, in the peripheral blood, miR-142-3p expression was significantly higher in the acute tubular necrosis group compared to the acute rejection group (P<0.05). Urine samples of the acute rejection group presented higher expression compared to the stable patients group (P<0.001) but the difference between acute tubular necrosis and acute rejection groups was not significant in the urinary analyzes (P=0.079). miR-142-3p expression has a distinct pattern of expression in the setting of post-operative acute tubular necrosis after kidney transplantation and may potentially be used as a non-invasive biomarker for renal graft dysfunction.

Co-transplantation of plasmid-transfected myoblasts and myotubes into rat brains enables high levels of gene expression long-term

NASA Technical Reports Server (NTRS)

Jiao, S.; Williams, P.; Safda, N.; Schultz, E.; Wolff, J. A.

1993-01-01

We have previously proposed the use of primary muscle cells as a "platform," or "vehicle" for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat brains for at least 6 mo without any decrease in graft size, or tumor formation. Stable, but moderate levels of intracerebral transgene expression were obtained by transplanting plasmid-transfected myotubes in culture. In the present study, high and stable levels of intracerebral transgene expression were achieved by the co-transplantation of plasmid-transfected myoblasts and myotubes in culture. Approximately 5 X 10(5) myoblasts and myotubes were transfected with 10 micrograms pRSVL plasmid DNA, and 30 micrograms Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, and much higher than the levels in myotube grafts, or co-grafts of myoblasts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains transplanted with co-graft of myoblasts, and myotubes transfected with pRSVLac-Z were stained immunofluorescently for beta-galactosidase activity. The muscle grafts contained beta-galactosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expression after postnatal gene transfer have rarely been achieved. Primary muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.

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

PubMed Central

2010-01-01

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

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

PubMed

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

2010-05-21

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

Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti.

PubMed

Kokoza, V; Ahmed, A; Cho, W L; Jasinskiene, N; James, A A; Raikhel, A

2000-08-01

Progress in molecular genetics makes possible the development of alternative disease control strategies that target the competence of mosquitoes to transmit pathogens. We tested the regulatory region of the vitellogenin (Vg) gene of Aedes aegypti for its ability to express potential antipathogen factors in transgenic mosquitoes. Hermes-mediated transformation was used to integrate a 2.1-kb Vg-promoter fragment driving the expression of the Defensin A (DefA) coding region, one of the major insect immune factors. PCR amplification of genomic DNA and Southern blot analyses, carried out through the ninth generation, showed that the Vg-DefA transgene insertion was stable. The Vg-DefA transgene was strongly activated in the fat body by a blood meal. The mRNA levels reached a maximum at 24-h postblood meal, corresponding to the peak expression time of the endogenous Vg gene. High levels of transgenic defensin were accumulated in the hemolymph of bloodfed female mosquitoes, persisting for 20-22 days after a single blood feeding. Purified transgenic defensin showed antibacterial activity comparable to that of defensin isolated from bacterially challenged control mosquitoes. Thus, we have been able to engineer the genetically stable transgenic mosquito with an element of systemic immunity, which is activated through the blood meal-triggered cascade rather than by infection. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in pathogen transmission.

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

PubMed

Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

2011-12-01

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors

NASA Astrophysics Data System (ADS)

Roizman, Bernard

1996-10-01

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.

PubMed

Hommais, Florence; Zghidi-Abouzid, Ouafa; Oger-Desfeux, Christine; Pineau-Chapelle, Emilie; Van Gijsegem, Frederique; Nasser, William; Reverchon, Sylvie

2011-01-01

Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted.

PubMed

Beinfeld, Margery C; Vishnuvardhan, Daesety; Blum, Alissa; Reynolds, Nicole; Fannous, Sanya; Kitagawa, Kouki; Marchand, James E

2006-04-01

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

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

Rosas, Maria F.; Vieira, Yuri A.; Postigo, Raul

2008-10-10

The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing eithermore » 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle.« less

Variation analysis of transcriptome changes reveals cochlear genes and their associated functions in cochlear susceptibility to acoustic overstimulation.

PubMed

Yang, Shuzhi; Cai, Qunfeng; Bard, Jonathan; Jamison, Jennifer; Wang, Jianmin; Yang, Weiping; Hu, Bo Hua

2015-12-01

Individual variation in the susceptibility of the auditory system to acoustic overstimulation has been well-documented at both the functional and structural levels. However, the molecular mechanism responsible for this variation is unclear. The current investigation was designed to examine the variation patterns of cochlear gene expression using RNA-seq data and to identify the genes with expression variation that increased following acoustic trauma. This study revealed that the constitutive expressions of cochlear genes displayed diverse levels of gene-specific variation. These variation patterns were altered by acoustic trauma; approximately one-third of the examined genes displayed marked increases in their expression variation. Bioinformatics analyses revealed that the genes that exhibited increased variation were functionally related to cell death, biomolecule metabolism, and membrane function. In contrast, the stable genes were primarily related to basic cellular processes, including protein and macromolecular syntheses and transport. There was no functional overlap between the stable and variable genes. Importantly, we demonstrated that glutamate metabolism is related to the variation in the functional response of the cochlea to acoustic overstimulation. Taken together, the results indicate that our analyses of the individual variations in transcriptome changes of cochlear genes provide important information for the identification of genes that potentially contribute to the generation of individual variation in cochlear responses to acoustic overstimulation. Copyright © 2015 Elsevier B.V. All rights reserved.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

PubMed

Norzagaray-Valenzuela, Claudia D; Germán-Báez, Lourdes J; Valdez-Flores, Marco A; Hernández-Verdugo, Sergio; Shelton, Luke M; Valdez-Ortiz, Angel

2018-05-16

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD 600  = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

PubMed Central

Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

2016-01-01

Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

PubMed Central

Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

PubMed

Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

Highly-efficient liposome-mediated transformation system for the basidiomycetous fungus Flammulina velutipes.

PubMed

Shi, Liang; Chen, Dongdong; Xu, Chao; Ren, Ang; Yu, Hanshou; Zhao, Mingwen

2017-07-11

Flammulina velutipes is a well-known edible mushroom cultivated all over the world. However, because of the low transformation frequency, the expensive instruments required, and the complicated, time-consuming procedures necessary, there is insufficient genetic research on F. velutipes. In this study, we report a liposome-mediated transformation (LMT) system for the genetic transformation of F. velutipes. Using the LMT system, we obtained 82 ± 4 stable F. velutipes transformants per 10 5 protoplasts, which is a clear increase in transformation frequency compared to the other methods used. We were able to detect the expression of an EGFP reporter gene in the F. velutipes transformants using fluorescence imaging assays. Furthermore, we used this method to transfer the laccase gene into F. velutipes and found that the transcriptional level and enzymatic activity increased in these transformants. Mitotic stability analysis showed that all of the selected transformants remained mitotically stable, even after five successive rounds of sub-culturing. These results demonstrate a new transgenic approach that will facilitate F. velutipes research.

Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals.

PubMed

Irie, T; Honda, Y; Hirano, T; Sato, T; Enei, H; Watanabe, T; Kuwahara, M

2001-09-01

It was reported that Pleurotus ostreatus was transformed unstably using recombinant plasmids containing a hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans expression signals, and that the plasmids were maintained extrachromosomally in the transformants. Here we report a stable and integrative transformation of the fungus to hygromycin B resistance, using a recombinant hph fused with Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase expression signals. Restriction-enzyme-mediated integration (REMI) was also tried and increased the transformation efficiency about ten-fold.

Gap-metric-based robustness analysis of nonlinear systems with full and partial feedback linearisation

NASA Astrophysics Data System (ADS)

Al-Gburi, A.; Freeman, C. T.; French, M. C.

2018-06-01

This paper uses gap metric analysis to derive robustness and performance margins for feedback linearising controllers. Distinct from previous robustness analysis, it incorporates the case of output unstructured uncertainties, and is shown to yield general stability conditions which can be applied to both stable and unstable plants. It then expands on existing feedback linearising control schemes by introducing a more general robust feedback linearising control design which classifies the system nonlinearity into stable and unstable components and cancels only the unstable plant nonlinearities. This is done in order to preserve the stabilising action of the inherently stabilising nonlinearities. Robustness and performance margins are derived for this control scheme, and are expressed in terms of bounds on the plant nonlinearities and the accuracy of the cancellation of the unstable plant nonlinearity by the controller. Case studies then confirm reduced conservatism compared with standard methods.

Power laws, discontinuities and regional city size distributions

USGS Publications Warehouse

Garmestani, A.S.; Allen, Craig R.; Gallagher, C.M.

2008-01-01

Urban systems are manifestations of human adaptation to the natural environment. City size distributions are the expression of hierarchical processes acting upon urban systems. In this paper, we test the entire city size distributions for the southeastern and southwestern United States (1990), as well as the size classes in these regions for power law behavior. We interpret the differences in the size of the regional city size distributions as the manifestation of variable growth dynamics dependent upon city size. Size classes in the city size distributions are snapshots of stable states within urban systems in flux.

Evaluation of RNA from human trabecular bone and identification of stable reference genes.

PubMed

Cepollaro, Simona; Della Bella, Elena; de Biase, Dario; Visani, Michela; Fini, Milena

2018-06-01

The isolation of good quality RNA from tissues is an essential prerequisite for gene expression analysis to study pathophysiological processes. This study evaluated the RNA isolated from human trabecular bone and defined a set of stable reference genes. After pulverization, RNA was extracted with a phenol/chloroform method and then purified using silica columns. The A260/280 ratio, A260/230 ratio, RIN, and ribosomal ratio were measured to evaluate RNA quality and integrity. Moreover, the expression of six candidates was analyzed by qPCR and different algorithms were applied to assess reference gene stability. A good purity and quality of RNA was achieved according to A260/280 and A260/230 ratios, and RIN values. TBP, YWHAZ, and PGK1 were the most stable reference genes that should be used for gene expression analysis. In summary, the method proposed is suitable for gene expression evaluation in human bone and a set of reliable reference genes has been identified. © 2017 Wiley Periodicals, Inc.

Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

PubMed

Yang, Zhimin; Chen, Yu; Hu, Baoyun; Tan, Zhiqun; Huang, Bingru

2015-01-01

Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

Comparative Cold Shock Expression and Characterization of Fungal Dye-Decolorizing Peroxidases.

PubMed

Behrens, Christoph J; Zelena, Kateryna; Berger, Ralf G

2016-08-01

Dye-decolorizing peroxidases (DyPs) from Auricularia auricula-judae, Bjerkandera adusta, Pleurotus ostreatus and Marasmius scorodonius (Basidiomycota) were expressed in Escherichia coli using the cold shock-inducible expression system pCOLD I DNA. Functional expression was achieved without the addition of hemin or the co-expression of any chaperones. The presence or absence of the native signal sequence had a strong impact on the success of the expression, but the effect was not consistent for the different DyPs. While BaDyP and AajDyP were stable at 50 °C, the more thermolabile MsP2 and PoDyp, upon catalytic intervention, lend themselves to more rapid thermal inactivation. The bleaching of norbixin (E 160b) using MsP2 was most efficient at pH 4.0, while BaDyP and AajDypP worked best in the weakly acidic to neutral range, indicating a choice of DyPs for a broad field of applications in different food matrices.

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster.

PubMed

Cantera, Rafael; Lüer, Karin; Rusten, Tor Erik; Barrio, Rosa; Kafatos, Fotis C; Technau, Gerhard M

2002-12-01

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.

Efficient production of artificially designed gelatins with a Bacillus brevis system.

PubMed

Kajino, T; Takahashi, H; Hirai, M; Yamada, Y

2000-01-01

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

PubMed Central

Sharma, Nynne; Cai, Yujia; Bak, Rasmus O; Jakobsen, Martin R; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

2013-01-01

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system. PMID:23443502

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.

Molecular Phenotypes Distinguish Patients with Relatively Stable from Progressive Idiopathic Pulmonary Fibrosis (IPF)

PubMed Central

Boon, Kathy; Bailey, Nathaniel W.; Yang, Jun; Steel, Mark P.; Groshong, Steve; Kervitsky, Dolly; Brown, Kevin K.; Schwarz, Marvin I.; Schwartz, David A.

2009-01-01

Background Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic interstitial lung disease that is unresponsive to current therapy and often leads to death. However, the rate of disease progression differs among patients. We hypothesized that comparing the gene expression profiles between patients with stable disease and those in which the disease progressed rapidly will lead to biomarker discovery and contribute to the understanding of disease pathogenesis. Methodology and Principal Findings To begin to address this hypothesis, we applied Serial Analysis of Gene Expression (SAGE) to generate lung expression profiles from diagnostic surgical lung biopsies in 6 individuals with relatively stable (or slowly progressive) IPF and 6 individuals with progressive IPF (based on changes in DLCO and FVC over 12 months). Our results indicate that this comprehensive lung IPF SAGE transcriptome is distinct from normal lung tissue and other chronic lung diseases. To identify candidate markers of disease progression, we compared the IPF SAGE profiles in stable and progressive disease, and identified a set of 102 transcripts that were at least 5-fold up regulated and a set of 89 transcripts that were at least 5-fold down regulated in the progressive group (P-value≤0.05). The over expressed genes included surfactant protein A1, two members of the MAPK-EGR-1-HSP70 pathway that regulate cigarette-smoke induced inflammation, and Plunc (palate, lung and nasal epithelium associated), a gene not previously implicated in IPF. Interestingly, 26 of the up regulated genes are also increased in lung adenocarcinomas and have low or no expression in normal lung tissue. More importantly, we defined a SAGE molecular expression signature of 134 transcripts that sufficiently distinguished relatively stable from progressive IPF. Conclusions These findings indicate that molecular signatures from lung parenchyma at the time of diagnosis could prove helpful in predicting the likelihood of disease progression or possibly understanding the biological activity of IPF. PMID:19347046

An Effective 3D Ear Acquisition System

PubMed Central

Liu, Yahui; Lu, Guangming; Zhang, David

2015-01-01

The human ear is a new feature in biometrics that has several merits over the more common face, fingerprint and iris biometrics. It can be easily captured from a distance without a fully cooperative subject. Also, the ear has a relatively stable structure that does not change much with the age and facial expressions. In this paper, we present a novel method of 3D ear acquisition system by using triangulation imaging principle, and the experiment results show that this design is efficient and can be used for ear recognition. PMID:26061553

An Effective 3D Ear Acquisition System.

PubMed

Liu, Yahui; Lu, Guangming; Zhang, David

2015-01-01

The human ear is a new feature in biometrics that has several merits over the more common face, fingerprint and iris biometrics. It can be easily captured from a distance without a fully cooperative subject. Also, the ear has a relatively stable structure that does not change much with the age and facial expressions. In this paper, we present a novel method of 3D ear acquisition system by using triangulation imaging principle, and the experiment results show that this design is efficient and can be used for ear recognition.

GENERATION OF TWO STABLE CELL LINES THAT EXPRESS HER-ALPHA OR HER-ALPHA AND -BETA AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING

EPA Science Inventory

Generation of Two Stable Cell Lines that Express hERa or
hERa and b and Firefly Luciferase Genes for Endocrine Screening

K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1

1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reprod...

Differentiation of human SH-SY5Y neuroblastoma cells by all-trans retinoic acid activates the interleukin-18 system.

PubMed

Sallmon, Hannes; Hoene, Victoria; Weber, Sven C; Dame, Christof

2010-02-01

The clinical prognosis of children with high-stage neuroblastoma is still poor. Therapeutic approaches include surgery and cellular differentiation by retinoic acid, but also experimental interleukin-based immune modulation. However, the molecular mechanisms of all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells are incompletely understood. Herein, we examined the effect of ATRA on the activity of the interleukin-18 (IL-18) system in human SH-SY5Y neuroblastoma cells. It is shown that SH-SY5Y cells express IL-18 receptor (IL-18R) and the secreted antagonist IL-18-binding protein (IL-18BP), but no IL-18. SH-SY5Y cells are highly sensitive to ATRA treatment and react by cellular differentiation from a neuroblastic toward a more neuronal phenotype. This was associated with induction of IL-18 and reduction of IL-18BP expression, while IL-18R expression remained stable. Thereby, we identified the IL-18 system as a novel target of ATRA in neuroblastoma cells that might contribute to the therapeutic properties of retinoids in treatment of neuroblastoma.

Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.

PubMed

Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

2016-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

Leptospiral outer membrane protein LipL41 is not essential for acute leptospirosis but requires a small chaperone protein, lep, for stable expression.

PubMed

King, Amy M; Bartpho, Thanatchaporn; Sermswan, Rasana W; Bulach, Dieter M; Eshghi, Azad; Picardeau, Mathieu; Adler, Ben; Murray, Gerald L

2013-08-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

PubMed

Tang, Danming; Lam, Cynthia; Louie, Salina; Hoi, Kam Hon; Shaw, David; Yim, Mandy; Snedecor, Brad; Misaghi, Shahram

2018-01-01

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genomic DNA-based absolute quantification of gene expression in Vitis

USDA-ARS?s Scientific Manuscript database

Many studies in which gene expression is quantified by polymerase chain reaction represent the expression of a gene of interest (GOI) relative to that of a reference gene (RG). Relative expression is founded on the assumptions that RG expression is stable across samples, treatments, organs, etc., an...

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

PubMed Central

Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Park, Ji-Sung; Lee, Seung-Chan; Baregundi Subbarao, Raghavendra; Lee, Sung-Lim; Park, Bong-Wook; King, William Allan; Rho, Gyu-Jin

2015-01-01

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs. PMID:25972899

Instability of Helios-deficient Tregs is associated with conversion to a T-effector phenotype and enhanced antitumor immunity.

PubMed

Nakagawa, Hidetoshi; Sido, Jessica M; Reyes, Edwin E; Kiers, Valerie; Cantor, Harvey; Kim, Hye-Jung

2016-05-31

Expression of the transcription factor Helios by Tregs ensures stable expression of a suppressive and anergic phenotype in the face of intense inflammatory responses, whereas Helios-deficient Tregs display diminished lineage stability, reduced FoxP3 expression, and production of proinflammatory cytokines. Here we report that selective Helios deficiency within CD4 Tregs leads to enhanced antitumor immunity through induction of an unstable phenotype and conversion of intratumoral Tregs into T effector cells within the tumor microenvironment. Induction of an unstable Treg phenotype is associated with enhanced production of proinflammatory cytokines by tumor-infiltrating but not systemic Tregs and significantly delayed tumor growth. Ab-dependent engagement of Treg surface receptors that result in Helios down-regulation also promotes conversion of intratumoral but not systemic Tregs into T effector cells and leads to enhanced antitumor immunity. These findings suggest that selective instability and conversion of intratumoral CD4 Tregs through genetic or Ab-based targeting of Helios may represent an effective approach to immunotherapy.

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

PubMed

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

2015-11-01

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

Iron-titanium oxyhydroxides which transport water into the deep upper mantle and mantle transition zone

NASA Astrophysics Data System (ADS)

Matsukage, K. N.; Nishihara, Y.

2015-12-01

We experimentally discovered a new hydrous phase in the system FeOOH-TiO2 at pressures of 10-16 GPa and temperatures of 1000-1600°C which corresponds to conditions of the deep upper mantle and the Earth's mantle transition zone. Seven different compositions in the FeOOH-TiO2 system having molar ratios of x = Ti/(Fe + Ti) = 0, 0.125, 0.25, 0.375, 0.5, 0.75 that were prepared by mixing reagent grade a-FeOOH (goethite) and TiO2 (anatase) powders were used as starting materials. High-pressure and high-temperature experiments were carried out using Kawai-type multi-anvil apparatus (Orange-1000 at Ehime University and SPI-1000 at Tokyo Institute of Technology). In this system, we identified two stable iron-titanium oxyhydroxide phases whose estimated composition is expressed by (FeH)1 - xTixO2 . One is the Fe-rich solid solution (x < 0.23) with e-FeOOH type crystal structure (e-phase, orthorhombic, P21nm) that was described by the previous studies (e.g., Suzuki 2010), and the other is the more Ti-rich solid solution (x > 0.35) with a-PbO2 type structure (a-phase, orthorhombic, Pbcn). The a-phase is stable up to 1500ºC for a composition of x = 0.5 and at least to 1600ºC for x = 0.75. Our result means that this phase is stable at average mantle temperature in the Earth's mantle transition zone. The Iron-titanium-rich hydrous phases was possible to stable in basalt + H2O system (e.g., Hashimoto and Matsukage 2013). Therefore our findings suggest that water transport in the Earth's deep interior is probably much more efficient than had been previously thought.

Regulation of EMMPRIN (CD147) on monocyte subsets in patients with symptomatic coronary artery disease.

PubMed

Sturhan, Henrik; Ungern-Sternberg, Saskia N I v; Langer, Harald; Gawaz, Meinrad; Geisler, Tobias; May, Andreas E; Seizer, Peter

2015-06-01

The role of individual monocyte subsets in inflammatory cardiovascular diseases is insufficiently understood. Although the Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) regulates important processes for inflammation such as MMP-release, its expression and regulation on monocyte subsets has not been characterized. In this clinical study, blood was obtained from 80 patients with stable coronary artery disease (CAD), 49 with acute myocardial infarction (AMI) and 34 healthy controls. Monocytes were divided into 3 subsets: CD14(++)CD16(-) (low), CD14(++)CD16(+) (intermediate), CD14(+)CD16(++) (high) according to phenotypic markers analyzed by flow cytometry. Surface expression of EMMPRIN was evaluated and compared with CD36 and CD47 expression. In all patients, EMMPRIN expression was significantly different among monocyte subsets with the highest expression on "classical" CD14(++)CD16(-) monocytes. EMMPRIN was upregulated on all monocyte subsets in patients with AMI as compared to patients with stable CAD. Notably, neither CD47 nor CD36 revealed a significant difference in patients with AMI compared to patients with stable CAD. EMMPRIN could serve as a marker for classical monocytes, which is upregulated in patients with acute myocardial infarction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Improved process conditions for increasing expression of MHC class II protein from a stable Drosophila S2 cell line.

PubMed

Shen, Xiao; Dojcinovic, Danijel; Baldi, Lucia; Hacker, David L; Luescher, Immanuel F; Wurm, Florian M

2018-01-01

To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR1 2xHis ) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

Cloning, Expression, and Biochemical Characterization of an Enantioselective Lipase, YLIP9, from Yarrowia lipolytica MSR80.

PubMed

Syal, Poonam; Gupta, Rani

2015-05-01

A novel lipase gene, ylip9, of Yarrowia lipolytica MSR80 was cloned and expressed in pEZZ18-HB101 system and was 99% identical to YLIP9 of Y. lipolytica CLIB122. It was purified using IgG-Sepharose as ZZ fused YLIP9 and had specific activity of 0.8 U/mg. ZZ-YLIP9 was most active at pH 8.0 and 70 °C. It was stable over a wide pH range of 3.0-11.0 and 100 % active at 70 °C up to 2 h and had t1/2 of 286.42 min at 80 °C. It showed high specificity toward p-nitrophenyldecanoate with kcat and catalytic efficiency of 30.17 s(-1) and 16.67 mM(-1) s(-1), respectively. It was non-regioselective, but an S-enantioselective lipase and the percentage conversion were enhanced in presence of hexane. ZZ-YLIP9 was stable in all of the organic solvents used, and its activity was enhanced by solvents having logP value less than 2.

Linking Low-Level Stable Isotope Fractionation to Expression of the Cytochrome P450 Monooxygenase-Encoding ethB Gene for Elucidation of Methyl tert-Butyl Ether Biodegradation in Aerated Treatment Pond Systems▿ †

PubMed Central

Jechalke, Sven; Rosell, Mònica; Martínez-Lavanchy, Paula M.; Pérez-Leiva, Paola; Rohwerder, Thore; Vogt, Carsten; Richnow, Hans H.

2011-01-01

Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [ɛC] of −0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [ɛH]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon (ɛC of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (ɛH of −5‰ ± 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem. PMID:21148686

Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae

PubMed Central

2013-01-01

Background Coffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease (CBD). This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling. Results In this study, a set of ten genes (S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and β-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72 hpi) between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes (resistant and susceptible), separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected. Conclusion Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae. PMID:24073624

C9ORF72 hexanucleotide repeat exerts toxicity in a stable, inducible motor neuronal cell model, which is rescued by partial depletion of Pten

PubMed Central

Stopford, Matthew J.; Higginbottom, Adrian; Hautbergue, Guillaume M.; Cooper-Knock, Johnathan; Mulcahy, Padraig J.; De Vos, Kurt J.; Renton, Alan E.; Pliner, Hannah; Calvo, Andrea; Chio, Adriano; Traynor, Bryan J.; Azzouz, Mimoun; Heath, Paul R.; Kirby, Janine

2017-01-01

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease, characterised by progressive failure of the neuromuscular system. A (G4C2)n repeat expansion in C9ORF72 is the most common genetic cause of ALS and frontotemporal dementia (FTD). To date, the balance of evidence indicates that the (G4C2)n repeat causes toxicity and neurodegeneration via a gain-of-toxic function mechanism; either through direct RNA toxicity or through the production of toxic aggregating dipeptide repeat proteins. Here, we have generated a stable and isogenic motor neuronal NSC34 cell model with inducible expression of a (G4C2)102 repeat, to investigate the gain-of-toxic function mechanisms. The expression of the (G4C2)102 repeat produces RNA foci and also undergoes RAN translation. In addition, the expression of the (G4C2)102 repeat shows cellular toxicity. Through comparison of transcriptomic data from the cellular model with laser-captured spinal motor neurons from C9ORF72-ALS cases, we also demonstrate that the PI3K/Akt cell survival signalling pathway is dysregulated in both systems. Furthermore, partial knockdown of Pten rescues the toxicity observed in the NSC34 (G4C2)102 cellular gain-of-toxic function model of C9ORF72-ALS. Our data indicate that PTEN may provide a potential therapeutic target to ameliorate toxic effects of the (G4C2)n repeat. PMID:28158451

Decreased RECQL5 correlated with disease progression of osteosarcoma

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

Wu, Junlong; Zhi, Liqiang; Dai, Xin

Human RecQ helicase family, consisting of RECQL, RECQL4, RECQL5, BLM and WRN, has critical roles in genetic stability and tumorigenesis. Although RECQL5 has been reported to correlate with the susceptibility to malignances including osteosarcoma, the specific effect on tumor genesis and progression is not yet clarified. Here we focused on the relationship between RECQL5 expression and osteosarcoma disease progression, and further investigated the function of RECQL5 on MG-63 cell proliferation and apoptosis. By immunohistochemical analysis, qRT-PCR and western blot, we found that RECQL5 expression was downregulated in osteosarcoma tissues and cells. Patients with advanced tumor stage and low grade expressedmore » lower RECQL5. To construct a stable RECQL5 overexpression osteosarcoma cell line (MG-63-RECQL5), RECQL5 gene was inserted into the human AAVS1 safe harbor by CRISPR/Cas9 system. The overexpression of RECQL5 was verified by qRT-PCR and western blot. Cell proliferation, cell cycle and apoptosis assay revealed that RECQL5 overexpression inhibited proliferation, induced G1-phase arrest and promoted apoptosis in MG-63 cells. Collectively, our results suggested RECQL5 as a tumor suppressor in osteosarcoma and may be a potential therapeutic target for osteosarcoma treatment. - Highlights: • The expression of RECQL5 was downregulated in osteosarcoma tissues and cells. • Decreased RECQL5 correlated with osteosarcoma Enneking surgical classification. • We constructed a stable RECQL5 overexpression cell line by CRISPR/Cas9 system. • RECQL5 overexpression inhibited proliferation of MG-63 cells. • RECQL5 overexpression promoted apoptosis of MG-63 cells.« less

Identification of FOPDT and SOPDT process dynamics using closed loop test.

PubMed

Bajarangbali, Raghunath; Majhi, Somanath; Pandey, Saurabh

2014-07-01

In this paper, identification of stable and unstable first order, second order overdamped and underdamped process dynamics with time delay is presented. Relay with hysteresis is used to induce a limit cycle output and using this information, unknown process model parameters are estimated. State space based generalized analytical expressions are derived to achieve accurate results. To show the performance of the proposed method expressions are also derived for systems with a zero. In real time systems, measurement noise is an important issue during identification of process dynamics. A relay with hysteresis reduces the effect of measurement noise, in addition a new multiloop control strategy is proposed to recover the original limit cycle. Simulation results are included to validate the effectiveness of the proposed method. Copyright © 2014 ISA. Published by Elsevier Ltd. All rights reserved.

From Saccharomyces cerevisiae to human: The important gene co-expression modules.

PubMed

Liu, Wei; Li, Li; Ye, Hua; Chen, Haiwei; Shen, Weibiao; Zhong, Yuexian; Tian, Tian; He, Huaqin

2017-08-01

Network-based systems biology has become an important method for analyzing high-throughput gene expression data and gene function mining. Yeast has long been a popular model organism for biomedical research. In the current study, a weighted gene co-expression network analysis algorithm was applied to construct a gene co-expression network in Saccharomyces cerevisiae . Seventeen stable gene co-expression modules were detected from 2,814 S. cerevisiae microarray data. Further characterization of these modules with the Database for Annotation, Visualization and Integrated Discovery tool indicated that these modules were associated with certain biological processes, such as heat response, cell cycle, translational regulation, mitochondrion oxidative phosphorylation, amino acid metabolism and autophagy. Hub genes were also screened by intra-modular connectivity. Finally, the module conservation was evaluated in a human disease microarray dataset. Functional modules were identified in budding yeast, some of which are associated with patient survival. The current study provided a paradigm for single cell microorganisms and potentially other organisms.

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

PubMed Central

Jalili, Reza B.; Forouzandeh, Farshad; Rezakhanlou, Alireza Moeen; Hartwell, Ryan; Medina, Abelardo; Warnock, Garth L.; Larijani, Bagher; Ghahary, Aziz

2010-01-01

OBJECTIVE The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets. RESEARCH DESIGN AND METHODS Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO–expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice. RESULTS IDO-expressing grafts survived significantly longer than controls (41.2 ± 1.64 vs. 12.9 ± 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming. CONCLUSIONS Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts. PMID:20522587

Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

PubMed

Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

2016-01-01

In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.

Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein

PubMed Central

Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

2016-01-01

In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

PubMed Central

Marlow, Jeffrey J.; Skennerton, Connor T.; Li, Zhou; Chourey, Karuna; Hettich, Robert L.; Pan, Chongle; Orphan, Victoria J.

2016-01-01

Marine methane seep habitats represent an important control on the global flux of methane. Nucleotide-based meta-omics studies outline community-wide metabolic potential, but expression patterns of environmentally relevant proteins are poorly characterized. Proteomic stable isotope probing (proteomic SIP) provides additional information by characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities, offering enhanced detection through system-wide product integration. Here we applied proteomic SIP to 15NH4+ and CH4 amended seep sediment microcosms in an attempt to track protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 unique proteins were identified, 11% of which were 15N-labeled. Consistent with the dominant anaerobic oxidation of methane (AOM) activity commonly observed in anoxic seep sediments, proteins associated with sulfate reduction and reverse methanogenesis—including the ANME-2 associated methylenetetrahydromethanopterin reductase (Mer)—were all observed to be actively synthesized (15N-enriched). Conversely, proteins affiliated with putative aerobic sulfur-oxidizing epsilon- and gammaproteobacteria showed a marked decrease over time in our anoxic sediment incubations. The abundance and phylogenetic range of 15N-enriched methyl-coenzyme M reductase (Mcr) orthologs, many of which exhibited novel post-translational modifications, suggests that seep sediments provide niches for multiple organisms performing analogous metabolisms. In addition, 26 proteins of unknown function were consistently detected and actively expressed under conditions supporting AOM, suggesting that they play important roles in methane seep ecosystems. Stable isotope probing in environmental proteomics experiments provides a mechanism to determine protein durability and evaluate lineage-specific responses in complex microbial communities placed under environmentally relevant conditions. Our work here demonstrates the active synthesis of a metabolically specific minority of enzymes, revealing the surprising longevity of most proteins over the course of an extended incubation experiment in an established, slow-growing, methane-impacted environmental system. PMID:27199908

Warming Alters Expressions of Microbial Functional Genes Important to Ecosystem Functioning

DOE PAGES

Xue, Kai; Xie, Jianping; Zhou, Aifen; ...

2016-05-06

Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward moremore » C 4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C 4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming.« less

Warming Alters Expressions of Microbial Functional Genes Important to Ecosystem Functioning

PubMed Central

Xue, Kai; Xie, Jianping; Zhou, Aifen; Liu, Feifei; Li, Dejun; Wu, Liyou; Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Luo, Yiqi; Zhou, Jizhong

2016-01-01

Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward more C4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming. PMID:27199978

Warming Alters Expressions of Microbial Functional Genes Important to Ecosystem Functioning

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

Xue, Kai; Xie, Jianping; Zhou, Aifen

Soil microbial communities play critical roles in ecosystem functioning and are likely altered by climate warming. However, so far, little is known about effects of warming on microbial functional gene expressions. Here, we applied functional gene array (GeoChip 3.0) to analyze cDNA reversely transcribed from total RNA to assess expressed functional genes in active soil microbial communities after nine years of experimental warming in a tallgrass prairie. Our results showed that warming significantly altered the community wide gene expressions. Specifically, expressed genes for degrading more recalcitrant carbon were stimulated by warming, likely linked to the plant community shift toward moremore » C 4 species under warming and to decrease the long-term soil carbon stability. In addition, warming changed expressed genes in labile C degradation and N cycling in different directions (increase and decrease), possibly reflecting the dynamics of labile C and available N pools during sampling. However, the average abundances of expressed genes in phosphorus and sulfur cycling were all increased by warming, implying a stable trend of accelerated P and S processes which might be a mechanism to sustain higher plant growth. Furthermore, the expressed gene composition was closely related to both dynamic (e.g., soil moisture) and stable environmental attributes (e.g., C 4 leaf C or N content), indicating that RNA analyses could also capture certain stable trends in the long-term treatment. Overall, this study revealed the importance of elucidating functional gene expressions of soil microbial community in enhancing our understanding of ecosystem responses to warming.« less

Development of an Efficient Agrobacterium-Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

PubMed Central

Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

2013-01-01

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst non-transgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits. PMID:23832764

Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

PubMed

Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

2013-08-01

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

PubMed

Popławski, Piotr; Wiśniewski, Jacek R; Rijntjes, Eddy; Richards, Keith; Rybicka, Beata; Köhrle, Josef; Piekiełko-Witkowska, Agnieszka

2017-01-01

Type 1 iodothyronine deiodinase (DIO1) contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4) yielding of 3,5,3'-triiodothyronine (T3), a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2) that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2), enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2), sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10). DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression and changes in local availability of thyroid hormones might favor a shift from a differentiated to a more proliferation-prone state of cancer tissues and cell lines.

Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system

PubMed Central

Rijntjes, Eddy; Richards, Keith; Rybicka, Beata; Köhrle, Josef

2017-01-01

Type 1 iodothyronine deiodinase (DIO1) contributes to deiodination of 3,5,3’,5’-tetraiodo-L-thyronine (thyroxine, T4) yielding of 3,5,3’-triiodothyronine (T3), a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The ‘downregulated’ group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2) that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2), enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2), sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10). DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression and changes in local availability of thyroid hormones might favor a shift from a differentiated to a more proliferation-prone state of cancer tissues and cell lines. PMID:29272308

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

PubMed

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

2012-07-11

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

Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems.

PubMed

Luger, Anna-Luisa; Sauer, Benedikt; Lorenz, Nadja I; Engel, Anna L; Braun, Yannick; Voss, Martin; Harter, Patrick N; Steinbach, Joachim P; Ronellenfitsch, Michael W

2018-05-17

Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01⁻1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.

Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris

PubMed Central

2013-01-01

Background Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. Findings Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. Conclusion Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation. PMID:24083672

HDM2 promotes WIP1-mediated medulloblastoma growth

PubMed Central

Buss, Meghan C.; Read, Tracy-Ann; Schniederjan, Matthew J.; Gandhi, Khanjan; Castellino, Robert C.

2012-01-01

Medulloblastoma is the most common malignant childhood brain tumor. The protein phosphatase and oncogene WIP1 is over-expressed or amplified in a significant number of primary human medulloblastomas and cell lines. In the present study, we examine an important mechanism by which WIP1 promotes medulloblastoma growth using in vitro and in vivo models. Human cell lines and intracerebellar xenografted animal models were used to study the role of WIP1 and the major TP53 regulator, HDM2, in medulloblastoma growth. Stable expression of WIP1 enhances growth of TP53 wild-type medulloblastoma cells, compared with cells with stable expression of an empty-vector or mutant WIP1. In an animal model, WIP1 enhances proliferation and reduces the survival of immunodeficient mice bearing intracerebellar xenografted human medulloblastoma cells. Cells with increased WIP1 expression also exhibit increased expression of HDM2. HDM2 knockdown or treatment with the HDM2 inhibitor Nutlin-3a, the active enantomer of Nutlin-3, specifically inhibits the growth of medulloblastoma cells with increased WIP1 expression. Nutlin-3a does not affect growth of medulloblastoma cells with stable expression of an empty vector or of mutant WIP1. Knockdown of WIP1 or treatment with the WIP1 inhibitor CCT007093 results in increased phosphorylation of known WIP1 targets, reduced HDM2 expression, and reduced growth specifically in WIP1 wild-type and high-expressing medulloblastoma cells. Combined WIP1 and HDM2 inhibition is more effective than WIP1 inhibition alone in blocking growth of WIP1 high-expressing medulloblastoma cells. Our preclinical study supports a role for therapies that target WIP1 and HDM2 in the treatment of medulloblastoma. PMID:22379189

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

SLACK, JEFFREY, M.

Wood is a potential source for biofuels such as ethanol if it can be digested into sugars and fermented by yeast. Biomass derived from wood is a challenging substrate for ethanol production since it is made of lignin and cellulose which cannot be broken down easily into fermentable sugars. Some insects, and termites in particular, are specialized at using enzymes in their guts to digest wood into sugars. If termite gut enzymes could be made abundantly by a recombinant protein expression vector system, they could be applied to an industrial process to make biofuels from wood. In this study, amore » large cDNA library of relevant termite genes was made using termites fed a normal diet, or a diet with added lignin. A subtracted library yielded genes that were overexpressed in the presence of lignin. Termite gut enzyme genes were identified and cloned into recombinant insect viruses called baculoviruses. Using our PERLXpress system for protein expression, these termite gene recombinant baculoviruses were prepared and used to infect insect larvae, which then expressed abundant recombinant termite enzymes. Many of these expressed enzymes were prepared to very high purity, and the activities were studied in conjunction with collaborators at Purdue University. Recombinant termite enzymes expressed in caterpillars were shown to be able to release sugars from wood. Mixing different combinations of these enzymes increased the amount of sugars released from a model woody biomass substrate. The most economical, fastest and energy conserving way to prepare termite enzymes expressed by recombinant baculoviruses in caterpillars was by making crude liquid homogenates. Making enzymes stable in homogenates therefore was a priority. During the course of these studies, improvements were made to the recombinant baculovirus expression platform so that caterpillar-derived homogenates containing expressed termite enzymes would be more stable. These improvements in the baculoviruses included significantly reducing proteases and preventing blackening immune reactions that occur when caterpillars are homogenized. Proteases may degrade enzymes and immune reaction blackening may inactivate enzymes thus compromising the ability of these crude recombinant expressed termite enzyme preparations to release sugars. Commercial preparations of fungal enzymes currently are used to digest wood for ethanol production. We demonstrated in this study that termite enzymes could improve the efficiency of fungal enzyme cocktails. Although the economic feasibility of using caterpillar expressed termite enzymes alone to treat wood was not proven, this work points to the potential to combine C-PERLXpressed insect enzymes with industrial enzyme cocktails to boost their efficiency at treating wood for biofuels.« less

Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support.

PubMed

Giri, Shibashish; Bader, Augustinus

2014-09-01

Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. We established a conditional human fetal hepatocytes cell line with mesenchymal characteristics. Thus immortalization of human fetal hepatocytes cell line by telomerase biology offers a great challenge to examine basic biological mechanisms which are directly related to human and best cell source having unlimited population doubling for bioartificial support without any risk of replicative senescence and pathogenic risks.

Dynamics of testosterone concentration in male steppe lemmings (Lagurus lagurus) in the reproductive cycle reflects the species-specific mating system.

PubMed

Potapova, O F; Potapov, M A; Kondratyuk, E Yu; Evsikov, V I

2016-05-01

In the blood of male steppe lemmings, relatively low background levels of testosterone were detected, this is characteristic of a monogamous species. A significant increase in testosterone level, more expressed in sexually active males, was observed at the initial stage of formation of reproductive couples. Apparently, in the future, the couple will exist in a stable relationship, and, hence, the maintenance of a high testosterone level becomes excessive. The decrease in, and the relative "normalization" of, the hormone level during the existence of the pair, including raising of the young, promotes higher expression of the male paternal care of the offspring at the species level.

Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

Cancer.gov

The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic stem (ES) cells from accumulating DNA damage and has been linked to hematopoietic differentiation of ES cells. Satyendra Singh, Ph.D., a postdoctoral fellow working with Philipp Oberdoerffer, Ph.D., in CCR’s Laboratory of Receptor Biology and Gene Expression, and their colleagues set out to determine whether Sirt1 could play a similar protective role in adult HSPCs.

A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides.

PubMed

Lemaire, Géraldine; de Sousa, Georges; Rahmani, Roger

2004-12-15

A stable hepatoma cell line expressing the human pregnane X receptor (hPXR) and the cytochrome P4503A4 (CYP3A4) distal and proximal promoters plus the luciferase reporter gene was developed to assess the ability of several xenobiotic agents to induce CYP3A4 and CYP2B6. After selection for neomycin resistance, one clone, displaying high luciferase activity in response to rifampicin (RIF), was isolated and the stable expression of hPXR was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Dose-response curves were generated by treating these cells with increasing concentrations of RIF, phenobarbital (PB), clotrimazole (CLOT) or 5beta-pregnane-3,20-dione (5beta-PREGN). The effective concentrations for half maximal response (EC50) were determined for each of these compounds. RIF was the most effective compound, with maximal luciferase activity induced at 10 microM. The agonist activities of PXR-specific inducers measured using our stable model were consistent with those measured in transient transfectants. The abilities of organochlorine (OC), organophosphate (OP) and pyrethroid pesticides (PY) to activate hPXR were also assessed and found to be consistent with the abilities of these compounds to induce CYP3A4 and CYP2B6 in primary culture of human hepatocytes. These results suggest that CYP3A4 and CYP2B6 regulation through PXR activation by persistent pesticides may have an impact on the metabolism of xenobiotic agents and endogenous steroid hormones. Our model provides a useful tool for studying hPXR activation and for identifying agents capable of inducing CYP3A4 and CYP2B6.

Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis.

PubMed

Yang, Chunxiao; Li, Hui; Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; Chu, Dong

2015-01-01

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔCt method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions.

The role of breast-feeding in infant immune system: a systems perspective on the intestinal microbiome.

PubMed

Praveen, Paurush; Jordan, Ferenc; Priami, Corrado; Morine, Melissa J

2015-09-24

The human intestinal microbiota changes from being sparsely populated and variable to possessing a mature, adult-like stable microbiome during the first 2 years of life. This assembly process of the microbiota can lead to either negative or positive effects on health, depending on the colonization sequence and diet. An integrative study on the diet, the microbiota, and genomic activity at the transcriptomic level may give an insight into the role of diet in shaping the human/microbiome relationship. This study aims at better understanding the effects of microbial community and feeding mode (breast-fed and formula-fed) on the immune system, by comparing intestinal metagenomic and transcriptomic data from breast-fed and formula-fed babies. We re-analyzed a published metagenomics and host gene expression dataset from a systems biology perspective. Our results show that breast-fed samples co-express genes associated with immunological, metabolic, and biosynthetic activities. The diversity of the microbiota is higher in formula-fed than breast-fed infants, potentially reflecting the weaker dependence of infants on maternal microbiome. We mapped the microbial composition and the expression patterns for host systems and studied their relationship from a systems biology perspective, focusing on the differences. Our findings revealed that there is co-expression of more genes in breast-fed samples but lower microbial diversity compared to formula-fed. Applying network-based systems biology approach via enrichment of microbial species with host genes revealed the novel key relationships of the microbiota with immune and metabolic activity. This was supported statistically by data and literature.

A DNA replicon system for rapid high-level production of virus-like particles in plants.

PubMed

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles; Mason, Hugh

2009-07-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. (c) 2009 Wiley Periodicals, Inc.

A DNA replicon system for rapid high-level production of virus-like particles in plants

PubMed Central

Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles

2009-01-01

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low level antigen accumulation and long time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this paper, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within five days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without p19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. PMID:19309755

A Novel Ideal Radionuclide Imaging System for Non-invasively Cell Monitoring built on Baculovirus Backbone by Introducing Sleeping Beauty Transposon

PubMed Central

Lv, Jing; Pan, Yu; Ju, Huijun; Zhou, Jinxin; Cheng, Dengfeng; Shi, Hongcheng; Zhang, Yifan

2017-01-01

Sleeping Beauty (SB) transposon is an attractive tool in stable transgene integration both in vitro and in vivo; and we introduced SB transposon into recombinant sodium-iodide symporter baculovirus system (Bac-NIS system) to facilitate long-term expression of recombinant sodium-iodide symporter. In our study, two hybrid baculovirus systems (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were successfully constructed and used to infect U87 glioma cells. After G418 selection screening, the Bac-eGFP-SB-NeoR-U87 cells remained eGFP positive, at the 18th and 196th day post transfection (96.03 ± 0.21% and 97.43 ± 0.81%), while eGFP positive population declined significantly at 18 days in cells transfected with unmodified baculovirus construct. NIS gene expression by Bac-NIS-SB-NeoR-U87 cells was also maintained for 28 weeks as determined by radioiodine uptake assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot (WB) assay. When transplanted in mice, Bac-NIS-SB-NeoR-U87 cells also expressed NIS gene stably as monitored by SPECT imaging for 43 days until the tumor-bearing mice were sacrificed. Herein, we showed that incorporation of SB in Bac-NIS system (hybrid Bac-NIS-SB-NeoR) can achieve a long-term transgene expression and can improve radionuclide imaging in cell tracking and monitoring in vivo. PMID:28262785

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

PubMed

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

2017-07-15

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

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

PubMed

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

2013-08-01

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

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines.

PubMed

Okumura, Fumihiko; Kameda, Hiroyuki; Ojima, Takao; Hatakeyama, Shigetsugu

2010-05-07

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes.

PubMed

Burke, James M; Kincaid, Rodney P; Aloisio, Francesca; Welch, Nicole; Sullivan, Christopher S

2017-09-29

Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of Growth and Metastases in an Estrogen Independent Breast Cancer Cell by Vitamin D Compounds

DTIC Science & Technology

2001-08-01

Utilization of green fluorescent protein for the identification of metastasis in an in vivo breast cancer model system. In Preparation. REPRINTS OF ALL...phenotype. Utilizing the SUM-159PT cell line stably transfected with pEGFP-Ci (enhanced green fluorescent protein ) we have been able to successfully...accurately detected. To develop a model with enhanced resolution of micrometastases we created a stable cell line expressing green fluorescent protein

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage lambda Q- vector.

PubMed

Oh, Jeong Seok; Cho, Daechul; Park, Tai Hyun

2005-11-01

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage lambda system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage lambda vector with a Q(-) mutation was used to enhance the expression of the lambda system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h(-1) with 1.5 S(0) of the medium supply. The maximum productivity of the total beta-galactosidase was 16.3x10(6) U l(-1) h(-1), which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.

The Gibbs free energy of nukundamite (Cu3.38Fe0.62S4): A correction and implications for phase equilibria

USGS Publications Warehouse

Seal, R.R.; Inan, E.E.; Hemingway, B.S.

2001-01-01

The Gibbs free energy of formation of nukundamite (Cu3.38Fe0.62S4) was calculated from published experimental studies of the reaction 3.25 Cu3.38Fe0.62S4 + S2 = 11 CuS + 2 FeS2 in order to correct an erroneous expression in the published record. The correct expression describing the Gibbs free energy of formation (kJ???mol-1) of nukundamite relative to the elements and ideal S2 gas is ??fG?? nukundamite T(K) = -549.75 + 0.23242 T + 3.1284 T0.5, with an uncertainty of 0.6%. An evaluation of the phase equilibria of nukundamite with associated phases in the system Cu-Fe-S as a function of temperature and sulfur fugacity indicates that nukundamite is stable from 224 to 501??C at high sulfidation states. At its greatest extent, at 434??C, the stability field of nukundamite is only 0.4 log f(S2) units wide, which explains its rarity. Equilibria between nukundamite and bornite, which limit the stability of both phases, involve bornite compositions that deviate significantly from stoichiometric Cu5FeS4. Under equilibrium conditions in the system Cu-Fe-S, nukundamite + chalcopyrite is not a stable assemblage at any temperature.

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

PubMed

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

2013-12-01

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

Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

PubMed Central

Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

2014-01-01

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes. PMID:25271645

Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

PubMed

Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

2014-01-01

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.

A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products.

PubMed

Haut, Larissa H; Gill, Amanda L; Kurupati, Raj K; Bian, Ang; Li, Yan; Giles-Davis, Wynetta; Xiang, Zhiquan; Zhou, Xiang Yang; Ertl, Hildegund C J

2016-10-01

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.

Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.

PubMed

Mardanov, Andrey V; Strakhova, Taisia S; Smagin, Vladimir A; Ravin, Nikolai V

2007-06-15

A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

PubMed

Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

2011-10-01

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

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

Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression.more » Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.« less

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

PubMed

Ferreira, Joshua P; Peacock, Ryan W S; Lawhorn, Ingrid E B; Wang, Clifford L

2011-12-01

The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

Construction of a recombinant wine yeast strain expressing beta-(1,4)-endoglucanase and its use in microvinification processes.

PubMed Central

Pérez-González, J A; González, R; Querol, A; Sendra, J; Ramón, D

1993-01-01

A genetic transformation system for an industrial wine yeast strain is presented here. The system is based on the acquisition of cycloheximide resistance and is a direct adaptation of a previously published procedure for brewing yeasts (L. Del Pozo, D. Abarca, M. G. Claros, and A. Jiménez, Curr. Genet. 19:353-358, 1991). Transformants arose at an optimal frequency of 0.5 transformant per microgram of DNA, are stable in the absence of selective pressure, and produce wine in the same way as the untransformed industrial strain. By using this transformation protocol, a filamentous fungal beta-(1,4)-endoglucanase gene has been expressed in an industrial wine yeast under the control of the yeast actin gene promoter. Endoglucanolytic wine yeast secretes the fungal enzyme to the must, producing a wine with an increased fruity aroma. Images PMID:8215355

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

PubMed

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

2016-11-01

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

Estimation of Dynamic Systems for Gene Regulatory Networks from Dependent Time-Course Data.

PubMed

Kim, Yoonji; Kim, Jaejik

2018-06-15

Dynamic system consisting of ordinary differential equations (ODEs) is a well-known tool for describing dynamic nature of gene regulatory networks (GRNs), and the dynamic features of GRNs are usually captured through time-course gene expression data. Owing to high-throughput technologies, time-course gene expression data have complex structures such as heteroscedasticity, correlations between genes, and time dependence. Since gene experiments typically yield highly noisy data with small sample size, for a more accurate prediction of the dynamics, the complex structures should be taken into account in ODE models. Hence, this study proposes an ODE model considering such data structures and a fast and stable estimation method for the ODE parameters based on the generalized profiling approach with data smoothing techniques. The proposed method also provides statistical inference for the ODE estimator and it is applied to a zebrafish retina cell network.

An approach to the determination of aircraft handling qualities using pilot transfer functions

NASA Technical Reports Server (NTRS)

Adams, J. J.; Hatch, H. G., Jr.

1978-01-01

It was shown that a correlation exists between pilot-aircraft system closed-loop characteristics, determined by using analytical expressions for pilot response along with the analytical expression for the aircraft response, and pilot ratings obtained in many previous flight and simulation studies. Two different levels of preferred pilot response were used. These levels were: (1) a static gain and a second-order lag function with a lag time constant of 0.2 second; and (2) a static gain, a lead time constant of 1 second, and a 0.2-second lag time constant. If a system response with a pitch-angle time constant of 2.6 seconds and a stable oscillatory mode of motion with a period of 2.5 seconds could be achieved with the first-level pilot model, it was shown that the pilot rating will be satisfactory for that vehicle.

Increasing Type 1 Poliovirus Capsid Stability by Thermal Selection

PubMed Central

Adeyemi, Oluwapelumi O.; Nicol, Clare

2016-01-01

ABSTRACT Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunization programs using live-attenuated oral (OPV) and/or inactivated (IPV) PV vaccines have systematically reduced its spread and paved the way for eradication. Immunization will continue posteradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. They could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines that mimic the “empty” capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert into an alternative conformation unsuitable for vaccine purposes. Stabilized ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we selected thermally stable ECs of type 1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild-type (wt) virions. We have identified mutations on the capsid surface and in internal networks that are responsible for EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilized VLPs could be used as novel vaccines. IMPORTANCE Poliomyelitis is a highly infectious disease caused by PV and is on the verge of eradication. There are biosafety concerns about reintroduction of the disease from current vaccines that require live virus for production. Recombinantly expressed virus-like particles (VLPs) could address these inherent problems. However, the genome-free capsids (ECs) of wt PV are unstable and readily change antigenicity to a form not suitable as a vaccine. Here, we demonstrate that the ECs of type 1 PV can be stabilized by selecting heat-resistant viruses. Our data show that some capsid mutations stabilize the ECs and could be applied as candidates to synthesize stable VLPs as future genome-free poliovirus vaccines. PMID:27928008

Epigenetic regulation of serotype expression antagonizes transcriptome dynamics in Paramecium tetraurelia

PubMed Central

Cheaib, Miriam; Dehghani Amirabad, Azim; Nordström, Karl J. V.; Schulz, Marcel H.; Simon, Martin

2015-01-01

Phenotypic variation of a single genotype is achieved by alterations in gene expression patterns. Regulation of such alterations depends on their time scale, where short-time adaptations differ from permanently established gene expression patterns maintained by epigenetic mechanisms. In the ciliate Paramecium, serotypes were described for an epigenetically controlled gene expression pattern of an individual multigene family. Paradoxically, individual serotypes can be triggered in Paramecium by alternating environments but are then stabilized by epigenetic mechanisms, thus raising the question to which extend their expression follows environmental stimuli. To characterize environmental adaptation in the context of epigenetically controlled serotype expression, we used RNA-seq to characterize transcriptomes of serotype pure cultures. The resulting vegetative transcriptome resource is first analysed for genes involved in the adaptive response to the altered environment. Secondly, we identified groups of genes that do not follow the adaptive response but show co-regulation with the epigenetically controlled serotype system, suggesting that their gene expression pattern becomes manifested by similar mechanisms. In our experimental set-up, serotype expression and the entire group of co-regulated genes were stable among environmental changes and only heat-shock genes altered expression of these gene groups. The data suggest that the maintenance of these gene expression patterns in a lineage represents epigenetically controlled robustness counteracting short-time adaptation processes. PMID:26231545

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

PubMed

Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

2017-08-10

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

CD137+CD154− Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures

PubMed Central

Nowak, Anna; Lock, Dominik; Bacher, Petra; Hohnstein, Thordis; Vogt, Katrin; Gottfreund, Judith; Giehr, Pascal; Polansky, Julia K.; Sawitzki, Birgit; Kaiser, Andrew; Walter, Jörn; Scheffold, Alexander

2018-01-01

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154− expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro. PMID:29467769

CD137+CD154- Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures.

PubMed

Nowak, Anna; Lock, Dominik; Bacher, Petra; Hohnstein, Thordis; Vogt, Katrin; Gottfreund, Judith; Giehr, Pascal; Polansky, Julia K; Sawitzki, Birgit; Kaiser, Andrew; Walter, Jörn; Scheffold, Alexander

2018-01-01

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154- expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro .

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis).

PubMed

Pinheiro, T T; Nishimura, D S; De Nadai, F B; Figueira, A; Latado, R R

2015-12-28

Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

PubMed Central

Tang, Xiaoli; Wang, Hongyan; Shao, Chuyang; Shao, Hongbo

2015-01-01

Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. PMID:26581422

The stable Cr isotopic compositions of chondrites and silicate planetary reservoirs

NASA Astrophysics Data System (ADS)

Schoenberg, Ronny; Merdian, Alexandra; Holmden, Chris; Kleinhanns, Ilka C.; Haßler, Kathrin; Wille, Martin; Reitter, Elmar

2016-06-01

The depletion of chromium in Earth's mantle (∼2700 ppm) in comparison to chondrites (∼4400 ppm) indicates significant incorporation of chromium into the core during our planet's metal-silicate differentiation, assuming that there was no significant escape of the moderately volatile element chromium during the accretionary phase of Earth. Stable Cr isotope compositions - expressed as the ‰-difference in 53Cr/52Cr from the terrestrial reference material SRM979 (δ53/52CrSRM979 values) - of planetary silicate reservoirs might thus yield information about the conditions of planetary metal segregation processes when compared to chondrites. The stable Cr isotopic compositions of 7 carbonaceous chondrites, 11 ordinary chondrites, 5 HED achondrites and 2 martian meteorites determined by a double spike MC-ICP-MS method are within uncertainties indistinguishable from each other and from the previously determined δ53/52CrSRM979 value of -0.124 ± 0.101‰ for the igneous silicate Earth. Extensive quality tests support the accuracy of the stable Cr isotope determinations of various meteorites and terrestrial silicates reported here. The uniformity in stable Cr isotope compositions of samples from planetary silicate mantles and undifferentiated meteorites indicates that metal-silicate differentiation of Earth, Mars and the HED parent body did not cause measurable stable Cr isotope fractionation between these two reservoirs. Our results also imply that the accretionary disc, at least in the inner solar system, was homogeneous in its stable Cr isotopic composition and that potential volatility loss of chromium during accretion of the terrestrial planets was not accompanied by measurable stable isotopic fractionation. Small but reproducible variations in δ53/52CrSRM979 values of terrestrial magmatic rocks point to natural stable Cr isotope variations within Earth's silicate reservoirs. Further and more detailed studies are required to investigate whether silicate differentiation processes, such as partial mantle melting and crystal fractionation, can cause stable Cr isotopic fractionation on Earth and other planetary bodies.

Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

PubMed

Kruzel, Marian L; Actor, Jeffrey K; Zimecki, Michał; Wise, Jasen; Płoszaj, Paulina; Mirza, Shaper; Kruzel, Mark; Hwang, Shen-An; Ba, Xueqing; Boldogh, Istvan

2013-12-01

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects

PubMed Central

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2-ΔΔCt method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR. PMID:22938136

Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.

PubMed

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2(-ΔΔCt) method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.

Localization and developmental expression of two chicken host defense peptides: cathelicidin-2 and avian β-defensin 9.

PubMed

Cuperus, Tryntsje; van Dijk, Albert; Dwars, R Marius; Haagsman, Henk P

2016-08-01

In the first weeks of life young chickens are highly susceptible to infectious diseases due to immaturity of the immune system. Little is known about the expression of host defense peptides (HDPs) during this period. In this study we examined the expression pattern of two chicken HDPs, the cathelicidin CATH-2 and the β-defensin AvBD9 by immunohistochemistry in a set of organs from embryonic day 12 until four weeks posthatch. AvBD9 was predominantly found in enteroendocrine cells throughout the intestine, the first report of in vivo HDP expression in this cell type, and showed stable expression levels during development. CATH-2 was exclusively found in heterophils which decreased after hatch in most of the examined organs including spleen, bursa and small intestine. In the lung CATH-2 expression was biphasic and peaked at the first day posthatch. In short, CATH-2 and AvBD9 appear to be expressed in cell types strategically located to respond to infectious stimuli, suggesting these peptides play a role in embryonic and early posthatch defense. Copyright © 2016 Elsevier Ltd. All rights reserved.

The endobacterium of an arbuscular mycorrhizal fungus modulates the expression of its toxin-antitoxin systems during the life cycle of its host.

PubMed

Salvioli di Fossalunga, Alessandra; Lipuma, Justine; Venice, Francesco; Dupont, Laurence; Bonfante, Paola

2017-10-01

Arbuscular mycorrhizal fungi (AMF) are widespread root symbionts that perform important ecological services, such as improving plant nutrient and water acquisition. Some AMF from the Gigasporaceae family host a population of endobacteria, Candidatus Glomeribacter gigasporarum (Cagg). The analysis of the Cagg genome identified six putative toxin-antitoxin modules (TAs), consisting of pairs of stable toxins and unstable antitoxins that affect diverse physiological functions. Sequence analysis suggested that these TA modules were acquired by horizontal transfer. Gene expression patterns of two TAs (yoeB/yefM and chpB/chpS) changed during the fungal life cycle, with the expression during the pre-symbiotic phase higher than during the symbiosis with the plant host. The heterologous expression in Escherichia coli demonstrated the functionality only for the YoeB-YefM pair. On the basis of these observations, we speculate that TA modules might help Cagg adapt to its intracellular habitat, coordinating its proliferation with the physiological state of the AMF host.

Applications of lentiviral vectors in molecular imaging.

PubMed

Chatterjee, Sushmita; De, Abhijit

2014-06-01

Molecular imaging provides the ability of simultaneous visual and quantitative estimation of long term gene expression directly from living organisms. To reveal the kinetics of gene expression by imaging method, often sustained expression of the transgene is required. Lentiviral vectors have been extensively used over last fifteen years for delivery of a transgene in a wide variety of cell types. Lentiviral vectors have the well known advantages such as sustained transgene delivery through stable integration into the host genome, the capability of infecting non-dividing and dividing cells, broad tissue tropism, a reasonably large carrying capacity for delivering therapeutic and reporter gene combinations. Additionally, they do not express viral proteins during transduction, have a potentially safe integration site profile, and a relatively easy system for vector manipulation and infective viral particle production. As a result, lentiviral vector mediated therapeutic and imaging reporter gene delivery to various target organs holds promise for the future treatment. In this review, we have conducted a brief survey of important lentiviral vector developments in diverse biomedical fields including reproductive biology.

ROBUSTNESS OF SIGNALING GRADIENT IN DROSOPHILA WING IMAGINAL DISC

PubMed Central

Lei, Jinzhi; Wan, Frederic Y. M.; Lander, Arthur D.; Nie, Qing

2012-01-01

Quasi-stable gradients of signaling protein molecules (known as morphogens or ligands) bound to cell receptors are known to be responsible for differential cell signaling and gene expressions. From these follow different stable cell fates and visually patterned tissues in biological development. Recent studies have shown that the relevant basic biological processes yield gradients that are sensitive to small changes in system characteristics (such as expression level of morphogens or receptors) or environmental conditions (such as temperature changes). Additional biological activities must play an important role in the high level of robustness observed in embryonic patterning for example. It is natural to attribute observed robustness to various type of feedback control mechanisms. However, our own simulation studies have shown that feedback control is neither necessary nor sufficient for robustness of the morphogen decapentaplegic (Dpp) gradient in wing imaginal disc of Drosophilas. Furthermore, robustness can be achieved by substantial binding of the signaling morphogen Dpp with nonsignaling cell surface bound molecules (such as heparan sulfate proteoglygans) and degrading the resulting complexes at a sufficiently rapid rate. The present work provides a theoretical basis for the results of our numerical simulation studies. PMID:24098092

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

PubMed Central

2012-01-01

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

Stable expression of calpain 3 from a muscle transgene in vivo: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation

PubMed Central

Spencer, M. J.; Guyon, J. R.; Sorimachi, H.; Potts, A.; Richard, I.; Herasse, M.; Chamberlain, J.; Dalkilic, I.; Kunkel, L. M.; Beckmann, J. S.

2002-01-01

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development. PMID:12084932

Selection of relatively exact reference genes for gene expression studies in goosegrass (Eleusine indica) under herbicide stress.

PubMed

Chen, Jingchao; Huang, Zhaofeng; Huang, Hongjuan; Wei, Shouhui; Liu, Yan; Jiang, Cuilan; Zhang, Jie; Zhang, Chaoxian

2017-04-21

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species.

Stable Reference Gene Selection for RT-qPCR Analysis in Nonviruliferous and Viruliferous Frankliniella occidentalis

PubMed Central

Pan, Huipeng; Ma, Yabin; Zhang, Deyong; Liu, Yong; Zhang, Zhanhong; Zheng, Changying; Chu, Dong

2015-01-01

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for measuring and evaluating gene expression during variable biological processes. To facilitate gene expression studies, normalization of genes of interest relative to stable reference genes is crucial. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), the main vector of tomato spotted wilt virus (TSWV), is a destructive invasive species. In this study, the expression profiles of 11 candidate reference genes from nonviruliferous and viruliferous F. occidentalis were investigated. Five distinct algorithms, geNorm, NormFinder, BestKeeper, the ΔC t method, and RefFinder, were used to determine the performance of these genes. geNorm, NormFinder, BestKeeper, and RefFinder identified heat shock protein 70 (HSP70), heat shock protein 60 (HSP60), elongation factor 1 α, and ribosomal protein l32 (RPL32) as the most stable reference genes, and the ΔC t method identified HSP60, HSP70, RPL32, and heat shock protein 90 as the most stable reference genes. Additionally, two reference genes were sufficient for reliable normalization in nonviruliferous and viruliferous F. occidentalis. This work provides a foundation for investigating the molecular mechanisms of TSWV and F. occidentalis interactions. PMID:26244556

Vitamin E-coated dialyzer membranes downregulate expression of monocyte adhesion and co-stimulatory molecules.

PubMed

Betjes, Michiel G H; Hoekstra, Franciska M E; Klepper, M; Postma, Saskia M; Vaessen, Leonard M B

2004-01-01

In patients on chronic hemodialysis leukocyte activation has been related to the impaired function of the immune system. In this study we investigated if the vitamin E-coated dialyzer membrane could reduce monocyte activation thereby improving cellular immunity. This hypothesis was tested in a prospective crossover trial in which 14 stable hemodialysis patients were switched from the baseline hemophane dialyzer to a vitamin E-coated and thereafter a polysulphone dialyzer membrane or vice versa. Monocyte MHC class I, CD54 and ICAM-1 expression was significantly downregulated when a vitamin E-coated or polysulphone dialyzer was used. The use of a vitamin E membrane specifically decreased monocyte CD40 and CD86 expression. Lectin induced T cell proliferation increased with the use of the vitamin E-coated membrane as compared to polysulphone and hemophane dialyzers. Vitamin E-coated dialyzers induced a less-activated phenotype of monocytes and may improve cellular immunity.

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Expression of recombinant antibacterial lactoferricin-related peptides from Pichia pastoris expression system.

PubMed

Chen, Gen-Hung; Chen, Wei-Ming; Huang, Guo-Ting; Chen, Yu-Wen; Jiang, Shann-Tzong

2009-10-28

Four recombinant antimicrobial peptide (rAMP) cDNAs, constructed from two goat lactoferricin-related peptide cDNAs (GLFcin and GLFcin II) with/without (His)(6)-Tag, were cloned into pPICZalphaC and transformed into Pichia pastoris SMD1168H. After methanol induction, these rAMPs were expressed and secreted into broth. They were purified after CM-Sepharose (without His-tg), HisTrap (with His-tg) and Sephadex G-25 chromatographies. The yield of purified rAMP was 0.15 mg/mL of broth. These 4 rAMPs were thermal-stable and with high antibacterial activity against Escherichia coli BCRC 11549, Pseudomonas aeruginosa BCRC 12450, Bacillus cereus BCRC 10603, Staphylococcus aureus BCRC 25923, Propioni bacterium acnes BCRC 10723, and Listera monocytogenes BCRC 14845. The minimum inhibitory concentration (MIC) of rAMPs against these indicators ranged from 4.07 to 16.00 mg/mL.

Short Alleles, Bigger Smiles? The Effect of 5-HTTLPR on Positive Emotional Expressions

PubMed Central

Haase, Claudia M.; Beermann, Ursula; Saslow, Laura R.; Shiota, Michelle N.; Saturn, Sarina R.; Lwi, Sandy J.; Casey, James J.; Nguyen, Nguyen K.; Whalen, Patrick K.; Keltner, Dacher J.; Levenson, Robert W.

2015-01-01

The present research examined the effect of the 5-HTTLPR polymorphism in the serotonin transporter gene on objectively coded positive emotional expressions (i.e., laughing and smiling behavior objectively coded using the Facial Action Coding System). Three studies with independent samples of participants were conducted. Study 1 examined young adults watching still cartoons. Study 2 examined young, middle-aged, and older adults watching a thematically ambiguous yet subtly amusing film clip. Study 3 examined middle-aged and older spouses discussing an area of marital conflict (which typically produces both positive and negative emotion). Aggregating data across studies, results showed that the short allele of 5-HTTLPR predicted heightened positive emotional expressions. Results remained stable when controlling for age, gender, ethnicity, and depressive symptoms. These findings are consistent with the notion that the short allele of 5-HTTLPR functions as an emotion amplifier, which may confer heightened susceptibility to environmental conditions. PMID:26029940

Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in Clostridium perfringens.

PubMed

Adachi, Keika; Ohtani, Kaori; Kawano, Michio; Singh, Ravindra Pal; Yousuf, Basit; Sonomoto, Kenji; Shimizu, Tohru; Nakayama, Jiro

2018-05-01

Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA) was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter, suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture, suggesting that organic acids produced by C. perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C. perfringens was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This suggested the possibility of pH-controlled anti-virulence strategies. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Structural Basis for Modulation of Quality Control Fate in a Marginally Stable Protein.

PubMed

Brock, Kelly P; Abraham, Ayelet-chen; Amen, Triana; Kaganovich, Daniel; England, Jeremy L

2015-07-07

The human von Hippel-Lindau (VHL) tumor suppressor is a marginally stable protein previously used as a model substrate of eukaryotic refolding and degradation pathways. When expressed in the absence of its cofactors, VHL cannot fold and is quickly degraded by the quality control machinery of the cell. We combined computational methods with in vivo experiments to examine the basis of the misfolding propensity of VHL. By expressing a set of randomly mutated VHL sequences in yeast, we discovered a more stable mutant form. Subsequent modeling suggested the mutation had caused a conformational change affecting cofactor and chaperone interaction, and this hypothesis was then confirmed by additional knockout and overexpression experiments targeting a yeast cofactor homolog. These findings offer a detailed structural basis for the modulation of quality control fate in a model misfolded protein and highlight burial mode modeling as a rapid means to detect functionally important conformational changes in marginally stable globular domains. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

PubMed

Sharapova, Olga A; Yurkova, Maria S; Fedorov, Alexey N

2016-02-01

We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Reference gene selection for normalization of RT-qPCR gene expression data from Actinidia deliciosa leaves infected with Pseudomonas syringae pv. actinidiae

PubMed Central

Petriccione, Milena; Mastrobuoni, Francesco; Zampella, Luigi; Scortichini, Marco

2015-01-01

Normalization of data, by choosing the appropriate reference genes (RGs), is fundamental for obtaining reliable results in reverse transcription-quantitative PCR (RT-qPCR). In this study, we assessed Actinidia deliciosa leaves inoculated with two doses of Pseudomonas syringae pv. actinidiae during a period of 13 days for the expression profile of nine candidate RGs. Their expression stability was calculated using four algorithms: geNorm, NormFinder, BestKeeper and the deltaCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protein phosphatase 2A (PP2A) were the most stable genes, while β-tubulin and 7s-globulin were the less stable. Expression analysis of three target genes, chosen for RGs validation, encoding the reactive oxygen species scavenging enzymes ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT) indicated that a combination of stable RGs, such as GAPDH and PP2A, can lead to an accurate quantification of the expression levels of such target genes. The APX level varied during the experiment time course and according to the inoculum doses, whereas both SOD and CAT resulted down-regulated during the first four days, and up-regulated afterwards, irrespective of inoculum dose. These results can be useful for better elucidating the molecular interaction in the A. deliciosa/P. s. pv. actinidiae pathosystem and for RGs selection in bacteria-plant pathosystems. PMID:26581656

[Evaluation on stability of internal controls in human cardiac muscle by real-time RT-PCR during early postmortem interval].

PubMed

Zhang, Ping; Ma, Kai-Jun; Zhang, Heng; Wang, Hui-Jun; Shen, Yi-Wen; Chen, Long

2012-04-01

To explore the stability of internal controls in human cardiac muscle by real-time RT-PCR during early postmortem interval (PMI) in order to find the most stable marker. Ten individuals with similar environmental conditions (the average store temperature: 25 degrees C) and different PMI ranging from 4.3 to 22.3 h were selected. Total RNA was extracted from each sample and six commonly internal controls were used including beta-actin, GAPDH, B2M, U6, 18S rRNA and HSA-miR-1, and the expression was detected in cardiac muscle by real-time RT-PCR. The expression stability of internal controls was evaluated using genormPLUS software during early PMI. The internal control with the most stability was selected. The relationship between the most stable marker and its expression level affected by some other parameters such as age, gender and cause of death was also analyzed. The U6 showed the most stable expression during early PMI in cardiac muscle, and its expression level was not affected by those parameters including age, gender and cause of death (P > 0.05). U6 may be a valuable internal control for the study of relationship between PMI determination and degradation of nucleic acid in human cardiac muscle by real-time RT-PCR.

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.

Impacts analysis of car following models considering variable vehicular gap policies

NASA Astrophysics Data System (ADS)

Xin, Qi; Yang, Nan; Fu, Rui; Yu, Shaowei; Shi, Zhongke

2018-07-01

Due to the important roles playing in the vehicles' adaptive cruise control system, variable vehicular gap polices were employed to full velocity difference model (FVDM) to investigate the traffic flow properties. In this paper, two new car following models were put forward by taking constant time headway(CTH) policy and variable time headway(VTH) policy into optimal velocity function, separately. By steady state analysis of the new models, an equivalent optimal velocity function was defined. To determine the linear stable conditions of the new models, we introduce equivalent expressions of safe vehicular gap, and then apply small amplitude perturbation analysis and long terms of wave expansion techniques to obtain the new models' linear stable conditions. Additionally, the first order approximate solutions of the new models were drawn at the stable region, by transforming the models into typical Burger's partial differential equations with reductive perturbation method. The FVDM based numerical simulations indicate that the variable vehicular gap polices with proper parameters directly contribute to the improvement of the traffic flows' stability and the avoidance of the unstable traffic phenomena.

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

PubMed

Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

2015-01-01

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. © 2015 by the Association of Clinical Scientists, Inc.

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

PubMed

Huang, Jinjin; Xia, Ji; Yang, Zhen; Guan, Feifei; Cui, Di; Guan, Guohua; Jiang, Wei; Li, Ying

2014-01-01

We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel. Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%. Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions.

PubMed

Saraiva, Kátia D C; Fernandes de Melo, Dirce; Morais, Vanessa D; Vasconcelos, Ilka M; Costa, José H

2014-09-01

The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

The Brassicaceae Family Displays Divergent, Shoot-Skewed NLR Resistance Gene Expression.

PubMed

Munch, David; Gupta, Vikas; Bachmann, Asger; Busch, Wolfgang; Kelly, Simon; Mun, Terry; Andersen, Stig Uggerhøj

2018-02-01

Nucleotide-binding site leucine-rich repeat resistance genes (NLRs) allow plants to detect microbial effectors. We hypothesized that NLR expression patterns could reflect organ-specific differences in effector challenge and tested this by carrying out a meta-analysis of expression data for 1,235 NLRs from nine plant species. We found stable NLR root/shoot expression ratios within species, suggesting organ-specific hardwiring of NLR expression patterns in anticipation of distinct challenges. Most monocot and dicot plant species preferentially expressed NLRs in roots. In contrast, Brassicaceae species, including oilseed rape ( Brassica napus ) and the model plant Arabidopsis ( Arabidopsis thaliana ), were unique in showing NLR expression skewed toward the shoot across multiple phylogenetically distinct groups of NLRs. The Brassicaceae are also outliers in the sense that they have lost the common symbiosis signaling pathway, which enables intracellular infection by root symbionts. While it is unclear if these two events are related, the NLR expression shift identified here suggests that the Brassicaceae may have evolved unique pattern-recognition receptors and antimicrobial root metabolites to substitute for NLR protection. Such innovations in root protection could potentially be exploited in crop rotation schemes or for enhancing root defense systems of non-Brassicaceae crops. © 2018 American Society of Plant Biologists. All Rights Reserved.

Efficient production of human acidic fibroblast growth factor in pea (Pisum sativum L.) plants by agroinfection of germinated seeds

PubMed Central

2011-01-01

Background For efficient and large scale production of recombinant proteins in plants transient expression by agroinfection has a number of advantages over stable transformation. Simple manipulation, rapid analysis and high expression efficiency are possible. In pea, Pisum sativum, a Virus Induced Gene Silencing System using the pea early browning virus has been converted into an efficient agroinfection system by converting the two RNA genomes of the virus into binary expression vectors for Agrobacterium transformation. Results By vacuum infiltration (0.08 Mpa, 1 min) of germinating pea seeds with 2-3 cm roots with Agrobacteria carrying the binary vectors, expression of the gene for Green Fluorescent Protein as marker and the gene for the human acidic fibroblast growth factor (aFGF) was obtained in 80% of the infiltrated developing seedlings. Maximal production of the recombinant proteins was achieved 12-15 days after infiltration. Conclusions Compared to the leaf injection method vacuum infiltration of germinated seeds is highly efficient allowing large scale production of plants transiently expressing recombinant proteins. The production cycle of plants for harvesting the recombinant protein was shortened from 30 days for leaf injection to 15 days by applying vacuum infiltration. The synthesized aFGF was purified by heparin-affinity chromatography and its mitogenic activity on NIH 3T3 cells confirmed to be similar to a commercial product. PMID:21548923

Animal production systems in the industrialised world.

PubMed

Sørensen, J T; Edwards, S; Noordhuizen, J; Gunnarsson, S

2006-08-01

The production of food from animal origin is relatively stable in the industrialised world. However, animal production systems are changing dramatically with respect to location, herd size and specialisation. Increased pressure from a critical public is moving animal-based production towards systems such as organic production and loose-housing systems which allow the animals to better express normal behaviour. The focus on food safety promotes systems with a high degree of biosecurity, often associated with an increase in herd size and self-containment. The globalisation of agricultural trade and increased competition also favours an increase in herd size and specialisation. These trends also lead to regions with livestock-dense areas, giving rise to environmental concerns. Therefore, good farming practice regulations and systems to provide a higher level of transparency, such as quality risk management programmes, are being developed.

The subtilisin-like protease SBT3 contributes to insect resistance in tomato

PubMed Central

Meyer, Michael; Huttenlocher, Franziska; Cedzich, Anna; Procopio, Susanne; Stroeder, Jasper; Pau-Roblot, Corinne; Lequart-Pillon, Michelle; Pelloux, Jérôme; Stintzi, Annick; Schaller, Andreas

2016-01-01

Subtilisin-like proteases (SBTs) constitute a large family of extracellular plant proteases, the function of which is still largely unknown. In tomato plants, the expression of SBT3 was found to be induced in response to wounding and insect attack in injured leaves but not in healthy systemic tissues. The time course of SBT3 induction resembled that of proteinase inhibitor II and other late wound response genes suggesting a role for SBT3 in herbivore defense. Consistent with such a role, larvae of the specialist herbivore Manduca sexta performed better on transgenic plants silenced for SBT3 expression (SBT3-SI). Supporting a contribution of SBT3 to systemic wound signaling, systemic induction of late wound response genes was attenuated in SBT3-SI plants. The partial loss of insect resistance may thus be explained by a reduction in systemic defense gene expression. Alternatively, SBT3 may play a post-ingestive role in plant defense. Similar to other anti-nutritive proteins, SBT3 was found to be stable and active in the insect’s digestive system, where it may act on unidentified proteins of insect or plant origin. Finally, a reduction in the level of pectin methylesterification that was observed in transgenic plants with altered levels of SBT3 expression suggested an involvement of SBT3 in the regulation of pectin methylesterases (PMEs). While such a role has been described in other systems, PME activity and the degree of pectin methylesterification did not correlate with the level of insect resistance in SBT3-SI and SBT3 overexpressing plants and are thus unrelated to the observed resistance phenotype. PMID:27259555

A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

PubMed

Rajendra, Yashas; Hougland, Maria D; Alam, Riazul; Morehead, Teresa A; Barnard, Gavin C

2015-05-01

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. © 2014 Wiley Periodicals, Inc.

Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs

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

Singh, Deepti; Rawat, Surender; Waseem, Mohd

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, K{sub m} values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative propertymore » against BSA and Cu{sup 2+}/H{sub 2}O{sub 2} model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies. - Highlights: • Laccase from Yersinia enterocolitica strain 7 was expressed in Escherichia coli BL21 (DE3). • Recombinant laccase was found to be thermostable and alkali tolerant. • The in silico and experimental studied proves the biotransformation of NSAIDs. • Laccase binds to ligands differentially under different protonation state. • Laccase also possesses free radical scavenging property.« less

Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

DOE PAGES

Marlow, Jeffery; Skennerton, Connor T.; Li, Zhou; ...

2016-04-29

Marine methane seep habitats represent an important control on the global flux of methane between the subsurface and water column reservoirs. Meta-omics studies have begun to outline community-wide metabolic potential, but expression patterns of proteins that enact sulfate-mediated anaerobic methane oxidation in seeps are poorly characterized. Proteomic stable isotope probing (proteomic SIP) offers an additional layer of information for characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities. Here we applied proteomic SIP to 15NH4+ and CH4 amended seep sediment microcosms in an attempt to track the protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 proteinsmore » were identified, 21% of which were 15N-labeled. We observed active synthesis (15N enrichment) of all proteins believed to be involved in sulfate reduction and reverse methanogenesis including methylenetetrahydromethanopterin reductase (Mer). The abundance and phylogenetic range of methyl-coenzyme M reductase (Mcr) orthologs produced during incubation experiments suggests that seeps provide sufficient niches for multiple organisms performing analogous metabolisms. Twenty-eight previously unreported post-translational modifications of McrA were measured, indicating dynamic enzymatic machinery and offering a dimension of functional diversity beyond gene-dictated sequence. RNA polymerase associated with putative sulfur-oxidizing Epsilonproteobacteria and aerobic Gammaproteobacteria were more abundant among pre-incubation proteins, suggesting diminished metabolic activity in long-term anoxic, sulfidic experimental incubations. Twenty-six proteins of unknown function were detected in all proteomic experiments and actively expressed in labeled experiments, suggesting that they play important roles in methane seep ecosystems. The addition of stable isotope probing to environmental proteomics experiments provides a mechanism to begin to assess the degree to which diagnostic meatbolic proteins are long-lived or acively synthesized in complex, slow-growing microbial communities. Our work here demonstrates that sediment-hosted microbial assemblages in marine methane seeps are dynamic, heterogeneous systems with broad functional diversity.« less

Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

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

Marlow, Jeffery; Skennerton, Connor T.; Li, Zhou

Marine methane seep habitats represent an important control on the global flux of methane between the subsurface and water column reservoirs. Meta-omics studies have begun to outline community-wide metabolic potential, but expression patterns of proteins that enact sulfate-mediated anaerobic methane oxidation in seeps are poorly characterized. Proteomic stable isotope probing (proteomic SIP) offers an additional layer of information for characterizing phylogenetically specific, functionally relevant activity in mixed microbial communities. Here we applied proteomic SIP to 15NH4+ and CH4 amended seep sediment microcosms in an attempt to track the protein synthesis of slow-growing, low-energy microbial systems. Across all samples, 3495 proteinsmore » were identified, 21% of which were 15N-labeled. We observed active synthesis (15N enrichment) of all proteins believed to be involved in sulfate reduction and reverse methanogenesis including methylenetetrahydromethanopterin reductase (Mer). The abundance and phylogenetic range of methyl-coenzyme M reductase (Mcr) orthologs produced during incubation experiments suggests that seeps provide sufficient niches for multiple organisms performing analogous metabolisms. Twenty-eight previously unreported post-translational modifications of McrA were measured, indicating dynamic enzymatic machinery and offering a dimension of functional diversity beyond gene-dictated sequence. RNA polymerase associated with putative sulfur-oxidizing Epsilonproteobacteria and aerobic Gammaproteobacteria were more abundant among pre-incubation proteins, suggesting diminished metabolic activity in long-term anoxic, sulfidic experimental incubations. Twenty-six proteins of unknown function were detected in all proteomic experiments and actively expressed in labeled experiments, suggesting that they play important roles in methane seep ecosystems. The addition of stable isotope probing to environmental proteomics experiments provides a mechanism to begin to assess the degree to which diagnostic meatbolic proteins are long-lived or acively synthesized in complex, slow-growing microbial communities. Our work here demonstrates that sediment-hosted microbial assemblages in marine methane seeps are dynamic, heterogeneous systems with broad functional diversity.« less

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

PubMed Central

Klessen, C; Schmidt, K H; Gumpert, J; Grosse, H H; Malke, H

1989-01-01

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates. Images PMID:2499253

Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

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

Lochter, A.; Galosy, S.; Muschler, J.

1997-08-11

Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, andmore » progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.« less

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.

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

PubMed

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

2011-12-01

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

A multi-landing pad DNA integration platform for mammalian cell engineering

PubMed Central

Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

2018-01-01

Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873

Epigenetic regulation of serotype expression antagonizes transcriptome dynamics in Paramecium tetraurelia.

PubMed

Cheaib, Miriam; Dehghani Amirabad, Azim; Nordström, Karl J V; Schulz, Marcel H; Simon, Martin

2015-08-01

Phenotypic variation of a single genotype is achieved by alterations in gene expression patterns. Regulation of such alterations depends on their time scale, where short-time adaptations differ from permanently established gene expression patterns maintained by epigenetic mechanisms. In the ciliate Paramecium, serotypes were described for an epigenetically controlled gene expression pattern of an individual multigene family. Paradoxically, individual serotypes can be triggered in Paramecium by alternating environments but are then stabilized by epigenetic mechanisms, thus raising the question to which extend their expression follows environmental stimuli. To characterize environmental adaptation in the context of epigenetically controlled serotype expression, we used RNA-seq to characterize transcriptomes of serotype pure cultures. The resulting vegetative transcriptome resource is first analysed for genes involved in the adaptive response to the altered environment. Secondly, we identified groups of genes that do not follow the adaptive response but show co-regulation with the epigenetically controlled serotype system, suggesting that their gene expression pattern becomes manifested by similar mechanisms. In our experimental set-up, serotype expression and the entire group of co-regulated genes were stable among environmental changes and only heat-shock genes altered expression of these gene groups. The data suggest that the maintenance of these gene expression patterns in a lineage represents epigenetically controlled robustness counteracting short-time adaptation processes. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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

PubMed

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

2012-10-01

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

Identification of Reference Genes for Quantitative Real-Time PCR in Date Palm (Phoenix dactylifera L.) Subjected to Drought and Salinity.

PubMed

V Patankar, Himanshu; M Assaha, Dekoum V; Al-Yahyai, Rashid; Sunkar, Ramanjulu; Yaish, Mahmoud W

2016-01-01

Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm's drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔCT method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm.

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

PubMed Central

Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses. PMID:26863232

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

PubMed

Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)

PubMed Central

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects. PMID:25356721

Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis (Thysanoptera: Thripidae).

PubMed

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease.

PubMed

Kaur, Punit; Nagaraja, Ganachari M; Zheng, Hongying; Gizachew, Dawit; Galukande, Moses; Krishnan, Sunil; Asea, Alexzander

2012-03-27

Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems. To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis. We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A). Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC.

A mouse model for triple-negative breast cancer tumor-initiating cells (TNBC-TICs) exhibits similar aggressive phenotype to the human disease

PubMed Central

2012-01-01

Background Triple-negative breast cancer (TNBC) exhibit characteristics quite distinct from other kinds of breast cancer, presenting as an aggressive disease--recurring and metastasizing more often than other kinds of breast cancer, without tumor-specific treatment options and accounts for 15% of all types of breast cancer with higher percentages in premenopausal African-American and Hispanic women. The reason for this aggressive phenotype is currently the focus of intensive research. However, progress is hampered by the lack of suitable TNBC cell model systems. Methods To understand the mechanistic basis for the aggressiveness of TNBC, we produced a stable TNBC cell line by sorting for 4T1 cells that do not express the estrogen receptor (ER), progesterone receptor (PgR) or the gene for human epidermal growth factor receptor 2 (HER2). As a control, we produced a stable triple-positive breast cancer (TPBC) cell line by transfecting 4T1 cells with rat HER2, ER and PgR genes and sorted for cells with high expression of ER and PgR by flow cytometry and high expression of the HER2 gene by Western blot analysis. Results We isolated tumor-initiating cells (TICs) by sorting for CD24+/CD44high/ALDH1+ cells from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) stable cell lines. Limiting dilution transplantation experiments revealed that CD24+/CD44high/ALDH1+ cells derived from TNBC (TNBC-TICs) and TPBC (TPBC-TICs) were significantly more effective at repopulating the mammary glands of naïve female BALB/c mice than CD24-/CD44-/ALDH1- cells. Implantation of the TNBC-TICs resulted in significantly larger tumors, which metastasized to the lungs to a significantly greater extent than TNBC, TPBC-TICs, TPBC or parental 4T1 cells. We further demonstrated that the increased aggressiveness of TNBC-TICs correlates with the presence of high levels of mouse twenty-five kDa heat shock protein (Hsp25/mouse HspB1) and seventy-two kDa heat shock protein (Hsp72/HspA1A). Conclusions Taken together, we have developed a TNBC-TICs model system based on the 4T1 cells which is a very useful metastasis model with the advantage of being able to be transplanted into immune competent recipients. Our data demonstrates that the TNBC-TICs model system could be a useful tool for studies on the pathogenesis and therapeutic treatment for TNBC. PMID:22452810

Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

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

Li, Jie; Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen; Yang, Xi-fei

2014-10-10

Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we exploremore » the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.« less

A plant-produced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with Bacillus anthracis Ames spores.

PubMed

Chichester, Jessica A; Manceva, Slobodanka D; Rhee, Amy; Coffin, Megan V; Musiychuk, Konstantin; Mett, Vadim; Shamloul, Moneim; Norikane, Joey; Streatfield, Stephen J; Yusibov, Vidadi

2013-03-01

The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our launch vector-based plant transient expression system. Using this system, we have successfully engineered, expressed, purified and characterized full-length PA (pp-PA83) in Nicotiana benthamiana plants using agroinfiltration. This plant-produced antigen elicited high toxin neutralizing antibody titers in mice and rabbits after two vaccine administrations with Alhydrogel. In addition, immunization with this vaccine candidate protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. The vaccine effects were dose-dependent and required the presence of Alhydrogel adjuvant. In addition, the vaccine antigen formulated with Alhydrogel was stable and retained immunogenicity after two-week storage at 4°C, the conditions intended for clinical use. These results support the testing of this vaccine candidate in human volunteers and the utility of our plant expression system for the production of a recombinant anthrax vaccine.

Development and characterization of transgenic dominant male sterile rice toward an outcross-based breeding system.

PubMed

Abe, Kiyomi; Oshima, Masao; Akasaka, Maiko; Konagaya, Ken-Ichi; Nanasato, Yoshihiko; Okuzaki, Ayako; Taniguchi, Yojiro; Tanaka, Junichi; Tabei, Yutaka

2018-03-01

Genomic selection is attracting attention in the field of crop breeding. To apply genomic selection effectively for autogamous (self-pollinating) crops, an efficient outcross system is desired. Since dominant male sterility is a powerful tool for easy and successive outcross of autogamous crops, we developed transgenic dominant male sterile rice ( Oryza sativa L.) using the barnase gene that is expressed by the tapetum-specific promoter BoA9 . Barnase -induced male sterile rice No. 10 (BMS10) was selected for its stable male sterility and normal growth characteristics. The BMS10 flowering habits, including heading date, flowering date, and daily flowering time of BMS10 tended to be delayed compared to wild type. When BMS10 and wild type were placed side-by-side and crossed under an open-pollinating condition, the seed-setting rate was <1.5%. When the clipping method was used to avoid the influence of late flowering habits, the seed-setting rate of BMS10 increased to a maximum of 86.4%. Although flowering synchronicity should be improved to increase the seed-setting rate, our results showed that this system can produce stable transgenic male sterility with normal female fertility in rice. The transgenic male sterile rice would promote a genomic selection-based breeding system in rice.

Prognostic factors of craniopharyngioma with special reference to autocrine/paracrine signaling: underestimated implication of growth hormone receptor.

PubMed

Ogawa, Yoshikazu; Watanabe, Mika; Tominaga, Teiji

2015-10-01

Craniopharyngioma is a slow-growing tumor classified as benign, but tight adhesion and significant local infiltration to the vital structures are common. In spite of improvement of modern microsurgery techniques and precise anatomical understanding not few cases of this tumor recur, and long-term tumor control and maintenance of quality of life are sometimes difficult. However, very little is known about the effects of the molecular characters of craniopharyngioma on the prognosis. Ninety eight cases of craniopharyngioma surgically treated at the Department of Neurosurgery, Tohoku University Hospital and Kohnan Hospital from April 1996 to May 2014, 45 males and 53 females aged from 2 to 80 years (mean, 40.84 years) were retrospectively reviewed, and postoperative outcomes and the possible involvement of the autocrine/paracrine mechanism were investigated. The patients were followed up at intervals of 6 months to assess tumor recurrence, and clinical outcomes were correlated with the findings of immunohistochemical examinations used growth hormone receptor (GHR) and downstream hormones. The follow-up period ranged from 3 to 209 months. Hormone expression was examined in 88 patients, of which 46 specimens (52.3 %) showed high expression of GHR. The GHR high expression group had a significantly shorter duration of postoperative stable disease compared with the low expression group (logrank test, p = 0.007). Simultaneous high expression of growth hormone (GH) and GHR was found in 33 specimens (37.5 %), and the high expression group had a significantly shorter duration of postoperative stable disease compared with the low expression group (logrank test, p = 0.011). No other hormones showed statistically significant differences in outcomes. High expression of GHR is associated with shorter duration of postoperative stable disease in patients with craniopharyngioma. If the surgical specimens were craniopharyngiomas with high GHR expression, GH supplementation would be introduced quite prudently.

Sexual stability in the nearly dioecious Pinus johannis (Pinaceae).

PubMed

Flores-Rentería, Lluvia; Molina-Freaner, Francisco; Whipple, Amy V; Gehring, Catherine A; Domínguez, C A

2013-03-01

Even though dioecy is a dominant sexual system among gymnosperms, little is known about its evolutionary history. Pinus johannis may represent a model system because unisexual and monoecious individuals compose its populations. The presence of unisexual individuals in other Pinus species is a consequence of sexual lability. Here we determined whether P. johannis represents the first example of a dioecious or nearly dioecious reproductive system in conifers by evaluating its sexual stability. • To assess the stability of sexual expression, we quantified the proportion of male vs. female reproductive structures produced by trees over multiple years and tested for the presence of sexual dimorphism. Sexual lability hypotheses were also examined by looking at the relationship between environmental factors and sexual expression and by comparing the reproductive behavior of P. johannis with its closest labile relative, P. edulis. • Pinus johannis is nearly dioecious: ~99% of individuals are unisexual or express a low proportion of the opposite gender with few changes in sexual expression through time. We found sexual dimorphism consistent with sexual stability. Sexual expression did not vary with tree size/age, abiotic environment, or herbivore removal, providing evidence against sexual lability. Individuals of P. johannis tended to produce only male or female strobili, whereas those of P. edulis were mainly monoecious with a gradient in the female to male strobili ratio. • This study represents the first report of a nearly stable dioecious Pinus species. The variety of sexual morphs coexisting in the same population makes P. johannis a model for studying the evolution of dioecy in gymnosperms.

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin*♦

PubMed Central

Samuel, Premila P.; Smith, Lucian P.; Phillips, George N.; Olson, John S.

2015-01-01

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. PMID:26205820

Exploring the nonlinear cloud and rain equation

NASA Astrophysics Data System (ADS)

Koren, Ilan; Tziperman, Eli; Feingold, Graham

2017-01-01

Marine stratocumulus cloud decks are regarded as the reflectors of the climate system, returning back to space a significant part of the income solar radiation, thus cooling the atmosphere. Such clouds can exist in two stable modes, open and closed cells, for a wide range of environmental conditions. This emergent behavior of the system, and its sensitivity to aerosol and environmental properties, is captured by a set of nonlinear equations. Here, using linear stability analysis, we express the transition from steady to a limit-cycle state analytically, showing how it depends on the model parameters. We show that the control of the droplet concentration (N), the environmental carrying-capacity (H0), and the cloud recovery parameter (τ) can be linked by a single nondimensional parameter (μ=√{N }/(ατH0) ) , suggesting that for deeper clouds the transition from open (oscillating) to closed (stable fixed point) cells will occur for higher droplet concentration (i.e., higher aerosol loading). The analytical calculations of the possible states, and how they are affected by changes in aerosol and the environmental variables, provide an enhanced understanding of the complex interactions of clouds and rain.

HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

PubMed

Gazi, Anastasia D; Charova, Spyridoula; Aivaliotis, Michalis; Panopoulos, Nicholas J; Kokkinidis, Michael

2015-01-01

Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evidence for a Stable Intermediate in Leukemia Virus Activation in AKR Mouse Embryo Cells

PubMed Central

Ihle, James N.; Kenney, Francis T.; Tennant, Raymond W.

1974-01-01

Analysis of the requirement for serum in the activation of the endogenous leukemia virus expression in AKR mouse embryo cells by 5-iododeoxyuridine shows that activation can be dissociated into two discrete serum-dependent events. The first involves incorporation of 5-iododeoxyuridine into DNA and results in the formation of a stable “activation intermediate” resembling the provirus formed during infection of stationary mouse embryo cells with exogenous leukemia virus. The second event, resulting in expression of the activation intermediate as synthesis of virus proteins, requires DNA replication but not 5-iododeoxyuridine. PMID:4604455

Selection of relatively exact reference genes for gene expression studies in goosegrass (Eleusine indica) under herbicide stress

PubMed Central

Chen, Jingchao; Huang, Zhaofeng; Huang, Hongjuan; Wei, Shouhui; Liu, Yan; Jiang, Cuilan; Zhang, Jie; Zhang, Chaoxian

2017-01-01

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species. PMID:28429727

Macrophage activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression.

PubMed

Blich, Miry; Golan, Amnon; Arvatz, Gil; Sebbag, Anat; Shafat, Itay; Sabo, Edmond; Cohen-Kaplan, Victoria; Petcherski, Sirouch; Avniel-Polak, Shani; Eitan, Amnon; Hammerman, Haim; Aronson, Doron; Axelman, Elena; Ilan, Neta; Nussbaum, Gabriel; Vlodavsky, Israel

2013-02-01

Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.

Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

PubMed Central

Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin

2016-01-01

Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels. PMID:27575051

Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1▿

PubMed Central

Zhan, Sien; Li, Jinming; Xu, Ruihuan; Wang, Lunan; Zhang, Kuo; Zhang, Rui

2009-01-01

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5′ terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4°C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA. PMID:19494069

Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1.

PubMed

Zhan, Sien; Li, Jinming; Xu, Ruihuan; Wang, Lunan; Zhang, Kuo; Zhang, Rui

2009-08-01

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNase-resistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5' terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4 degrees C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA.

Optimal harvesting of a stochastic delay logistic model with Lévy jumps

NASA Astrophysics Data System (ADS)

Qiu, Hong; Deng, Wenmin

2016-10-01

The optimal harvesting problem of a stochastic time delay logistic model with Lévy jumps is considered in this article. We first show that the model has a unique global positive solution and discuss the uniform boundedness of its pth moment with harvesting. Then we prove that the system is globally attractive and asymptotically stable in distribution under our assumptions. Furthermore, we obtain the existence of the optimal harvesting effort by the ergodic method, and then we give the explicit expression of the optimal harvesting policy and maximum yield.

Highly stable beta-class carbonic anhydrases useful in carbon capture systems

DOEpatents

Alvizo, Oscar; Benoit, Mike; Novick, Scott

2013-04-16

The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

Highly stable beta-class carbonic anhydrases useful in carbon capture systems

DOEpatents

Alvizo, Oscar; Benoit, Michael R; Novick, Scott J

2013-08-20

The present disclosure relates to .beta.-class carbonic anhydrase polypeptides having improved properties including increased thermostability and/or stability in the presence of amine compounds, ammonia, or carbonate ion. The present disclosure also provides formulations and uses of the polypeptides for accelerating the absorption of carbon dioxide from a gas stream into a solution as well as for the release of the absorbed carbon dioxide for further treatment and/or sequestering. Also provided are polynucleotides encoding the carbonic anhydrase polypeptides and host cells capable of expressing them.

Gene expression signature of tolerance and lymphocyte subsets in stable renal transplants: results of a cross-sectional study.

PubMed

Moreso, Francesc; Torres, Irina B; Martínez-Gallo, Monica; Benlloch, Susana; Cantarell, Carme; Perelló, Manel; Jimeno, José; Pujol-Borrell, Ricardo; Seron, Daniel

2014-06-01

In kidney transplants operational tolerance has been associated with up-regulation of B cell differentiation genes and an increased number of total, naive and transitional peripheral B cells. The aim is to evaluate tolerance biomarkers in different cohorts of stable renal transplants under immunosuppression. This is a cross-sectional study conducted in renal transplants. We evaluate genetic tolerance signature and lymphocyte subsets in stable transplants treated with calcineurin inhibitors (CNI) at 1 (n=15), 5 (n=14) and 10 (n=16) years, and azathioprine-treated transplants followed 30 years (n=8). Healthy volunteers (n=10) and patients with chronic rejection (n=15) served as controls. We confirm that peripheral expression of IGKV1D-13 and IGKV4-1 genes by RT-PCR distinguish tolerant (n=10) from stable transplants (n=10) provided by the International Tolerance Network. Tolerance signature was defined as the lowest expression for both genes in tolerant patients. In CNI-treated patients, genetic signature of tolerance and B cells showed a time-dependent increase not observed in azathioprine-treated patients (p<0.01). Genetic tolerance signature was observed in 0% at 1, 7% at 5 and 25% at 10-years while it was not observed in azathioprine-treated and chronic rejection patients. Fifteen out of 16 CNI-treated transplants at 10 years were revaluated 3 months apart. Nine did not show the tolerance signature in any determination, 4 in one and 2 in both determinations. Genetic signature of tolerance was associated with an increase of total, naive and transitional B cells (p<0.05). IGKV1D-13 and IGKV4-1 gene expression and its linked B cell populations increase during follow up in CNI-treated patients. At 10 years, 2 out of 15 CNI treated patients consistently express biomarkers associated with true tolerance. In azathioprine-treated patients these biomarkers were down-regulated. Copyright © 2014 Elsevier B.V. All rights reserved.

A Systems Biology Analysis Unfolds the Molecular Pathways and Networks of Two Proteobacteria in Spaceflight and Simulated Microgravity Conditions

NASA Astrophysics Data System (ADS)

Roy, Raktim; Phani Shilpa, P.; Bagh, Sangram

2016-09-01

Bacteria are important organisms for space missions due to their increased pathogenesis in microgravity that poses risks to the health of astronauts and for projected synthetic biology applications at the space station. We understand little about the effect, at the molecular systems level, of microgravity on bacteria, despite their significant incidence. In this study, we proposed a systems biology pipeline and performed an analysis on published gene expression data sets from multiple seminal studies on Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium under spaceflight and simulated microgravity conditions. By applying gene set enrichment analysis on the global gene expression data, we directly identified a large number of new, statistically significant cellular and metabolic pathways involved in response to microgravity. Alteration of metabolic pathways in microgravity has rarely been reported before, whereas in this analysis metabolic pathways are prevalent. Several of those pathways were found to be common across studies and species, indicating a common cellular response in microgravity. We clustered genes based on their expression patterns using consensus non-negative matrix factorization. The genes from different mathematically stable clusters showed protein-protein association networks with distinct biological functions, suggesting the plausible functional or regulatory network motifs in response to microgravity. The newly identified pathways and networks showed connection with increased survival of pathogens within macrophages, virulence, and antibiotic resistance in microgravity. Our work establishes a systems biology pipeline and provides an integrated insight into the effect of microgravity at the molecular systems level.

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

NASA Astrophysics Data System (ADS)

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

2018-06-01

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

A novel cell model to study the function of the adrenoleukodystrophy-related protein

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

Gueugnon, Fabien; Volodina, Natalia; Taouil, Jaoued Et

2006-03-03

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycyclinemore » dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural context.« less

Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals.

PubMed

Yu, Tao; Zhou, Yongjin J; Wenning, Leonie; Liu, Quanli; Krivoruchko, Anastasia; Siewers, Verena; Nielsen, Jens; David, Florian

2017-05-26

Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C 22 H 46 O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l -1 in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.

Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don.

PubMed

Xiao, Zheng; Sun, Xiaobo; Liu, Xiaoqing; Li, Chang; He, Lisi; Chen, Shangping; Su, Jiale

2016-01-01

The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that EF1- α (elongation factor 1-alpha), 18S (18s ribosomal RNA), and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin ( TUB ) was the least stable. ACT5 (actin), RPL3 , 18S , and EF1- α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle . Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1- α, 18S , ACT5 , RPL3 , and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle .

Quantum Kramers model: Corrections to the linear response theory for continuous bath spectrum

NASA Astrophysics Data System (ADS)

Rips, Ilya

2017-01-01

Decay of the metastable state is analyzed within the quantum Kramers model in the weak-to-intermediate dissipation regime. The decay kinetics in this regime is determined by energy exchange between the unstable mode and the stable modes of thermal bath. In our previous paper [Phys. Rev. A 42, 4427 (1990), 10.1103/PhysRevA.42.4427], Grabert's perturbative approach to well dynamics in the case of the discrete bath [Phys. Rev. Lett. 61, 1683 (1988), 10.1103/PhysRevLett.61.1683] has been extended to account for the second order terms in the classical equations of motion (EOM) for the stable modes. Account of the secular terms reduces EOM for the stable modes to those of the forced oscillator with the time-dependent frequency (TDF oscillator). Analytic expression for the characteristic function of energy loss of the unstable mode has been derived in terms of the generating function of the transition probabilities for the quantum forced TDF oscillator. In this paper, the approach is further developed and applied to the case of the continuous frequency spectrum of the bath. The spectral density functions of the bath of stable modes are expressed in terms of the dissipative properties (the friction function) of the original bath. They simplify considerably for the one-dimensional systems, when the density of phonon states is constant. Explicit expressions for the fourth order corrections to the linear response theory result for the characteristic function of the energy loss and its cumulants are obtained for the particular case of the cubic potential with Ohmic (Markovian) dissipation. The range of validity of the perturbative approach in this case is determined (γ /ωb<0.26 ), which includes the turnover region. The dominant correction to the linear response theory result is associated with the "work function" and leads to reduction of the average energy loss and its dispersion. This reduction increases with the increasing dissipation strength (up to ˜10 % ) within the range of validity of the approach. We have also calculated corrections to the depopulation factor and the escape rate for the quantum and for the classical Kramers models. Results for the classical escape rate are in very good agreement with the numerical simulations for high barriers. The results can serve as an additional proof of the robustness and accuracy of the linear response theory.

Quantum Kramers model: Corrections to the linear response theory for continuous bath spectrum.

PubMed

Rips, Ilya

2017-01-01

Decay of the metastable state is analyzed within the quantum Kramers model in the weak-to-intermediate dissipation regime. The decay kinetics in this regime is determined by energy exchange between the unstable mode and the stable modes of thermal bath. In our previous paper [Phys. Rev. A 42, 4427 (1990)PLRAAN1050-294710.1103/PhysRevA.42.4427], Grabert's perturbative approach to well dynamics in the case of the discrete bath [Phys. Rev. Lett. 61, 1683 (1988)PRLTAO0031-900710.1103/PhysRevLett.61.1683] has been extended to account for the second order terms in the classical equations of motion (EOM) for the stable modes. Account of the secular terms reduces EOM for the stable modes to those of the forced oscillator with the time-dependent frequency (TDF oscillator). Analytic expression for the characteristic function of energy loss of the unstable mode has been derived in terms of the generating function of the transition probabilities for the quantum forced TDF oscillator. In this paper, the approach is further developed and applied to the case of the continuous frequency spectrum of the bath. The spectral density functions of the bath of stable modes are expressed in terms of the dissipative properties (the friction function) of the original bath. They simplify considerably for the one-dimensional systems, when the density of phonon states is constant. Explicit expressions for the fourth order corrections to the linear response theory result for the characteristic function of the energy loss and its cumulants are obtained for the particular case of the cubic potential with Ohmic (Markovian) dissipation. The range of validity of the perturbative approach in this case is determined (γ/ω_{b}<0.26), which includes the turnover region. The dominant correction to the linear response theory result is associated with the "work function" and leads to reduction of the average energy loss and its dispersion. This reduction increases with the increasing dissipation strength (up to ∼10%) within the range of validity of the approach. We have also calculated corrections to the depopulation factor and the escape rate for the quantum and for the classical Kramers models. Results for the classical escape rate are in very good agreement with the numerical simulations for high barriers. The results can serve as an additional proof of the robustness and accuracy of the linear response theory.

Pt/Au nanoalloy supported on alumina and chlorided alumina: DFT and experimental analysis

NASA Astrophysics Data System (ADS)

Sharifi, N.; Falamaki, C.; Ghorbanzadeh Ahangari, M.

2018-04-01

Density functional theory (DFT) was used to explore the adsorption of Pt/Au nanoalloy onto a pure and chlorided γ-Al2O3(110) surface, which has been applied in numerous catalytic reactions. First, we considered the adsorption properties of Pt clusters (n ≤ 5) onto the Al2O3(110) surface to determine the most stable Pt cluster on alumina surface in reforming processes. After full structural relaxations of Pt clusters at various configurations on alumina, our computed results expressed that the minimum binding energy (‑5.67 eV) is accrued for Pt4 cluster and the distance between the nearest Pt atom in the cluster to the alumina surface is equal to 1.13 Å. Then, we investigated the binding energies, geometries, and electronic properties of adsorbed Aun clusters (n ≤ 6) on the γ-Al2O3(110) surface. Our studied showed that Au5 was the most thermodynamically stable structure on γ-Al2O3. Finally, we inspected these properties for adsorbed Au clusters onto the Pt4-decorated alumina (Aun/Pt4-alumina) system. The binding energy of the Au4/Pt4-alumina system was ‑5.01 eV, and the distance between Au4 cluster and Pt4-alumina was 1.33 Å. The Au4/Pt4alumina system was found to be the most stable nanometer-sized catalyst design. At last, our first-principles calculations predicted that the best position of embedment Cl on the Au4/Pt4-alumina.

Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

PubMed Central

2014-01-01

Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual. PMID:24884716

Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity.

PubMed

Refsland, Eric W; Hultquist, Judd F; Luengas, Elizabeth M; Ikeda, Terumasa; Shaban, Nadine M; Law, Emily K; Brown, William L; Reilly, Cavan; Emerman, Michael; Harris, Reuben S

2014-11-01

The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

[Establishment of an iRFP and luciferase dual-color fluorescence-traced hepatocellular carcinoma transplantation model in nude mice].

PubMed

Li, Hongjun; Yang, Tianhua; Huang, Yanping; Liu, Mingzhu; Qin, Zhongqiang; Chu, Fei; Li, Zhenghong; Li, Yonghai

2017-11-01

Objective To establish a hepatocellular carcinoma xenograft model in nude mice which could stably express gene and be monitored dynamically. Methods We first constructed the lentiviral particles containing luciferase (Luc) and near-infrared fluorescent protein (iRFP) and puromycin resistance gene, and then transduced them into the HepG2 hepatoma cells. The cell line stably expressing Luc and iRFP genes were screened and inoculated into nude mice to establish xenograft tumor model. Tumor growth was monitored using in vivo imaging system. HE staining and immunohistochemistry were used to evaluate the pathological features and tumorigenic ability. Results HepG2 cells stably expressing iRFP and Luc were obtained; with the engineered cell line, xenograft model was successfully established with the features of proper tumor developing time and high rate of tumor formation as well as typical pathological features as showed by HE staining and immunohistochemistry. Conclusion Hepatocellular carcinoma model in nude mice with the features of stable gene expression and dynamical monitoring has been established successfully with the HepG2-iRFP-Luc cell line.

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

PubMed Central

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

2009-01-01

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

Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

NASA Astrophysics Data System (ADS)

Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

1997-09-01

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

Quantifying facial expression signal and intensity use during development.

PubMed

Rodger, Helen; Lao, Junpeng; Caldara, Roberto

2018-06-12

Behavioral studies investigating facial expression recognition during development have applied various methods to establish by which age emotional expressions can be recognized. Most commonly, these methods employ static images of expressions at their highest intensity (apex) or morphed expressions of different intensities, but they have not previously been compared. Our aim was to (a) quantify the intensity and signal use for recognition of six emotional expressions from early childhood to adulthood and (b) compare both measures and assess their functional relationship to better understand the use of different measures across development. Using a psychophysical approach, we isolated the quantity of signal necessary to recognize an emotional expression at full intensity and the quantity of expression intensity (using neutral expression image morphs of varying intensities) necessary for each observer to recognize the six basic emotions while maintaining performance at 75%. Both measures revealed that fear and happiness were the most difficult and easiest expressions to recognize across age groups, respectively, a pattern already stable during early childhood. The quantity of signal and intensity needed to recognize sad, angry, disgust, and surprise expressions decreased with age. Using a Bayesian update procedure, we then reconstructed the response profiles for both measures. This analysis revealed that intensity and signal processing are similar only during adulthood and, therefore, cannot be straightforwardly compared during development. Altogether, our findings offer novel methodological and theoretical insights and tools for the investigation of the developing affective system. Copyright © 2018 Elsevier Inc. All rights reserved.

Commensal Streptococcus mitis is a unique vector for oral mucosal vaccination

PubMed Central

Daifalla, Nada; Cayabyab, Mark J.; Xie, Emily; Kim, Hyeun Bum; Tzipori, Saul; Stashenko, Philip; Duncan, Margaret; Campos-Neto, Antonio

2014-01-01

The development of vaccine approaches that induce mucosal and systemic immune responses is critical for the effective prevention of several infections. Here, we report on the use of the abundant human oral commensal bacterium Streptococcus mitis as a delivery vehicle for mucosal immunization. Using homologous recombination we generated a stable rS. mitis expressing a Mycobacterium tuberculosis protein (Ag85b). Oral administration of rS. mitis in gnotobiotic piglets resulted in efficient oral colonization and production of oral and systemic anti-Ag85b specific IgA and IgG antibodies. These results support that the commensal S. mitis is potentially a useful vector for mucosal vaccination. PMID:25522856

Inhibition of apoptosis by knockdown of caspase-3 with siRNA in rat bone marrow mesenchymal stem cells.

PubMed

Hua, Ping; Liu, Li-Bao; Liu, Jia-Liang; Wang, Meng; Jiang, Hui-Qi; Zeng, Kuan; Yang, Yan-Qi; Yang, Song-Ran

2013-09-01

Transplantation of bone marrow mesenchymal stem cells is a promising new strategy for the repair of infarcted cardiac tissue. However, the majority of transplanted bone marrow mesenchymal stem cells (BMSCs) die soon after transplantation, due in part to oxidative stress in the ischemic region. Oxidative stress is known to induce apoptosis through the activation of caspase-3. The aim of this study is to determine whether small interfering RNA targeting caspase-3 can inhibit the apoptosis of rat BMSCs in vitro. Caspase-3 siRNA expression vectors were prepared and transfected into rat BMSCs in the presence of liposomes. Western blot assay and real-time polymerase chain reaction (RT-PCR) were performed to detect caspase-3 expression. A retrovirus packaging system was employed to package 293FT cells producing caspase-3 siRNA virus, which were transfected into rat BMSCs. Those stably expressing caspase-3 siRNA were screened by Western blot assay and RT-PCR to determine caspase-3 expression levels. Stable transfection of caspase-3 siRNA significantly decreased caspase-3 protein (0.26 ± 0.001 vs. 0.42 ± 0.004, P < 0.05) and mRNA expression (0.19 ± 0.002 vs. 1, P < 0.05) in BMSCs compared to non-transfected BMSCs. Cells were incubated in H2O2 to induce apoptosis, which was detected by TUNEL staining, and BMSC morphology was not altered by either transient or stable transfection of caspase-3 siRNA. H2O2-induced apoptosis of BMSCs stably transfected with caspase-3 siRNA was dramatically reduced compared to that of normal BMSCs (11.0 ± 3.2 vs. 25.8 ± 4.2, P < 0.05). Caspase-3 knockdown BMSCs are thus more resistant to apoptosis than normal BMSCs, potentially increasing their survival rates under conditions that cause oxidative stress.

Enhanced Host-Parasite Resistance Based on Down-Regulation of Phelipanche aegyptiaca Target Genes Is Likely by Mobile Small RNA

PubMed Central

Dubey, Neeraj K.; Eizenberg, Hanan; Leibman, Diana; Wolf, Dalia; Edelstein, Menahem; Abu-Nassar, Jackline; Marzouk, Sally; Gal-On, Amit; Aly, Radi

2017-01-01

RNA silencing refers to diverse mechanisms that control gene expression at transcriptional and post-transcriptional levels which can also be used in parasitic pathogens of plants that Broomrapes (Orobanche/Phelipanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops and cause severe negative effects on the yield and yield quality of those crops. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we suggest an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipanche aegyptiaca genes PaACS, PaM6PR, and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing vector and stable expression in transgenic tomato Solanum lycopersicum (Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACS and PaM6PR transcripts from P. aegyptiaca tubercles grown on transgenic tomato or on TRV-infected Nicotiana benthamiana plants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamiana plants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes. PMID:28955363

Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies.

PubMed

Hoffmann, Stefan A; Müller-Vieira, Ursula; Biemel, Klaus; Knobeloch, Daniel; Heydel, Sandra; Lübberstedt, Marc; Nüssler, Andreas K; Andersson, Tommy B; Gerlach, Jörg C; Zeilinger, Katrin

2012-12-01

Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver. Copyright © 2012 Wiley Periodicals, Inc.

Exploring valid internal-control genes in Porphyra yezoensis (Bangiaceae) during stress response conditions

NASA Astrophysics Data System (ADS)

Wang, Wenlei; Wu, Xiaojie; Wang, Chao; Jia, Zhaojun; He, Linwen; Wei, Yifan; Niu, Jianfeng; Wang, Guangce

2014-07-01

To screen the stable expression genes related to the stress (strong light, dehydration and temperature shock) we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y(II) and F v/ F m were significantly affected when stress was imposed on the thalli of P orphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress (at salinity 90) on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-1α showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.

Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis.

PubMed

Fuentes, Alejandra; Ortiz, Javier; Saavedra, Nicolás; Salazar, Luis A; Meneses, Claudio; Arriagada, Cesar

2016-04-01

The gene expression stability of candidate reference genes in the roots and leaves of Solanum lycopersicum inoculated with arbuscular mycorrhizal fungi was investigated. Eight candidate reference genes including elongation factor 1 α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), protein phosphatase 2A (PP2Acs), ribosomal protein L2 (RPL2), β-tubulin (TUB), ubiquitin (UBI) and actin (ACT) were selected, and their expression stability was assessed to determine the most stable internal reference for quantitative PCR normalization in S. lycopersicum inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The stability of each gene was analysed in leaves and roots together and separated using the geNorm and NormFinder algorithms. Differences were detected between leaves and roots, varying among the best-ranked genes depending on the algorithm used and the tissue analysed. PGK, TUB and EF1 genes showed higher stability in roots, while EF1 and UBI had higher stability in leaves. Statistical algorithms indicated that the GAPDH gene was the least stable under the experimental conditions assayed. Then, we analysed the expression levels of the LePT4 gene, a phosphate transporter whose expression is induced by fungal colonization in host plant roots. No differences were observed when the most stable genes were used as reference genes. However, when GAPDH was used as the reference gene, we observed an overestimation of LePT4 expression. In summary, our results revealed that candidate reference genes present variable stability in S. lycopersicum arbuscular mycorrhizal symbiosis depending on the algorithm and tissue analysed. Thus, reference gene selection is an important issue for obtaining reliable results in gene expression quantification. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

PubMed

Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

2015-09-01

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression.

PubMed

Jiang, Xiaoou; Yu, Han; Teo, Cui Rong; Tan, Genim Siu Xian; Goh, Sok Chin; Patel, Parasvi; Chua, Yiqiang Kevin; Hameed, Nasirah Banu Sahul; Bertoletti, Antonio; Patzel, Volker

2016-09-01

Dumbbell-shaped DNA minimal vectors lacking nontherapeutic genes and bacterial sequences are considered a stable, safe alternative to viral, nonviral, and naked plasmid-based gene-transfer systems. We investigated novel molecular features of dumbbell vectors aiming to reduce vector size and to improve the expression of noncoding or coding RNA. We minimized small hairpin RNA (shRNA) or microRNA (miRNA) expressing dumbbell vectors in size down to 130 bp generating the smallest genetic expression vectors reported. This was achieved by using a minimal H1 promoter with integrated transcriptional terminator transcribing the RNA hairpin structure around the dumbbell loop. Such vectors were generated with high conversion yields using a novel protocol. Minimized shRNA-expressing dumbbells showed accelerated kinetics of delivery and transcription leading to enhanced gene silencing in human tissue culture cells. In primary human T cells, minimized miRNA-expressing dumbbells revealed higher stability and triggered stronger target gene suppression as compared with plasmids and miRNA mimics. Dumbbell-driven gene expression was enhanced up to 56- or 160-fold by implementation of an intron and the SV40 enhancer compared with control dumbbells or plasmids. Advanced dumbbell vectors may represent one option to close the gap between durable expression that is achievable with integrating viral vectors and short-term effects triggered by naked RNA.

Unstable genomes elevate transcriptome dynamics

PubMed Central

Stevens, Joshua B.; Liu, Guo; Abdallah, Batoul Y.; Horne, Steven D.; Ye, Karen J.; Bremer, Steven W.; Ye, Christine J.; Krawetz, Stephen A.; Heng, Henry H.

2015-01-01

The challenge of identifying common expression signatures in cancer is well known, however the reason behind this is largely unclear. Traditionally variation in expression signatures has been attributed to technological problems, however recent evidence suggests that chromosome instability (CIN) and resultant karyotypic heterogeneity may be a large contributing factor. Using a well-defined model of immortalization, we systematically compared the pattern of genome alteration and expression dynamics during somatic evolution. Co-measurement of global gene expression and karyotypic alteration throughout the immortalization process reveals that karyotype changes influence gene expression as major structural and numerical karyotypic alterations result in large gene expression deviation. Replicate samples from stages with stable genomes are more similar to each other than are replicate samples with karyotypic heterogeneity. Karyotypic and gene expression change during immortalization is dynamic as each stage of progression has a unique expression pattern. This was further verified by comparing global expression in two replicates grown in one flask with known karyotypes. Replicates with higher karyotypic instability were found to be less similar than replicates with stable karyotypes. This data illustrates the karyotype, transcriptome, and transcriptome determined pathways are in constant flux during somatic cellular evolution (particularly during the macroevolutionary phase) and this flux is an inextricable feature of CIN and essential for cancer formation. The findings presented here underscore the importance of understanding the evolutionary process of cancer in order to design improved treatment modalities. PMID:24122714

Gene Expression Signatures Based on Variability can Robustly Predict Tumor Progression and Prognosis

PubMed Central

Dinalankara, Wikum; Bravo, Héctor Corrada

2015-01-01

Gene expression signatures are commonly used to create cancer prognosis and diagnosis methods, yet only a small number of them are successfully deployed in the clinic since many fail to replicate performance on subsequent validation. A primary reason for this lack of reproducibility is the fact that these signatures attempt to model the highly variable and unstable genomic behavior of cancer. Our group recently introduced gene expression anti-profiles as a robust methodology to derive gene expression signatures based on the observation that while gene expression measurements are highly heterogeneous across tumors of a specific cancer type relative to the normal tissue, their degree of deviation from normal tissue expression in specific genes involved in tissue differentiation is a stable tumor mark that is reproducible across experiments and cancer types. Here we show that constructing gene expression signatures based on variability and the anti-profile approach yields classifiers capable of successfully distinguishing benign growths from cancerous growths based on deviation from normal expression. We then show that this same approach generates stable and reproducible signatures that predict probability of relapse and survival based on tumor gene expression. These results suggest that using the anti-profile framework for the discovery of genomic signatures is an avenue leading to the development of reproducible signatures suitable for adoption in clinical settings. PMID:26078586

Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate.

PubMed Central

Ryals, J; Little, R; Bremer, H

1982-01-01

The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments. PMID:6179924

Prognostic Role of Phospho-STAT3 in Patients with Cancers of the Digestive System: A Systematic Review and Meta-Analysis.

PubMed

Li, Mu-xing; Bi, Xin-yu; Huang, Zhen; Zhao, Jian-jun; Han, Yue; Li, Zhi-Yu; Zhang, Ye-fan; Li, Yuan; Chen, Xiao; Hu, Xu-hui; Zhao, Hong; Cai, Jian-qiang

2015-01-01

The definite prognostic role of p-STAT3 has not been well defined. We performed a meta-analysis evaluating the prognostic role of p-STAT3 expression in patients with digestive system cancers. We searched the available articles reporting the prognostic value of p-STAT3 in patients with cancers of the digestive system, mainly including colorectal cancer, gastric cancer, hepatocellular carcinoma, esophagus cancer and pancreatic cancer. The pooled hazard ratios (HRs) with 95 % confidence intervals (95 % CIs) of overall survival (OS) and disease-free survival (DFS) were used to assess the prognostic role of p-STAT3 expression level in cancer tissues. And the association between p-STAT3 expression and clinicopathological characteristics was evaluated. A total of 22 studies with 3585 patients were finally enrolled in the meta-analysis. The results showed that elevated p-STAT3 expression level predicted inferior OS (HR = 1.809, 95% CI: 1.442-2.270, P < 0.001) and DFS (HR = 1.481, 95% CI: 1.028-2.133, P = 0.035) in patients with malignant cancers of the digestive system. Increased expression of p-STAT3 is significantly related with tumor cell differentiation (Odds ratio (OR) = 1.895, 95% CI: 1.364-2.632, P < 0.001) and lymph node metastases (OR = 2.108, 95% CI: 1.104-4.024, P = 0.024). Sensitivity analysis suggested that the pooled HR was stable and omitting a single study did not change the significance of the pooled HR. Funnel plots and Egger's tests revealed there was no significant publication bias in the meta-analysis. Phospho-STAT3 might be a prognostic factor of patients with digestive system cancers. More well designed studies with adequate follow-up are needed to gain a thorough understanding of the prognostic role of p-STAT3.

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

PubMed

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

2016-01-01

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

Stable zymomonas mobilis xylose and arabinose fermenting strains

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Taipei, TW

2008-04-08

The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

[Early attachement relationships and epigenetic customization].

PubMed

Rocchi, Giordana; Serio, Valentina; Carluccio, Giuseppe Mattia; Marini, Isabella; Meuti, Valentina; Zaccagni, Michela; Giacchetti, Nicoletta; Aceti, Franca

2015-01-01

Recently, new findings in epigenetic science switched the focus from the observation of physiological intragenomic dynamics to the idea of an environmental co-construction of phenotypic expression. In psichodynamic field, objectual relations and attachement theoreticians emphasized the interpersonal dimension of individual development, focusing the attention on the relational matrix of self organization. The construction of stable affective-behavioral traits throughout different parenting styles has actually found a coincidence in ethological studies, which have explored the epigenetic processes underlying the relationship between caregiving and HPA stress responsiveness. An adequate parenting style seems to support affective regulation throughout psychobiological hidden moderators, which would tend to rebalance the physiological systems homeostasis; an unconfident attachment style would promote, on the other hand, the allostatic load rise. Sites of longlife epigenetic susceptibility have also been identified in humans; although associated with risk of maladaptive developing in adverse environmental conditions, they seem to confer protection under favorable conditions. This persisting possibility of reorganization of stable traits throughout lifetime, which seems to be activated by a relevant environmental input, grant to significant relationships, and to therapeutical one as well, an implicit reconditioning potential which could result into the configuration of new stable affective-behavioral styles.

Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation

NASA Astrophysics Data System (ADS)

Hay, Jake J.; Rodrigo-Navarro, Aleixandre; Hassi, Karoliina; Moulisova, Vladimira; Dalby, Matthew J.; Salmeron-Sanchez, Manuel

2016-02-01

Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.

Short alleles, bigger smiles? The effect of 5-HTTLPR on positive emotional expressions.

PubMed

Haase, Claudia M; Beermann, Ursula; Saslow, Laura R; Shiota, Michelle N; Saturn, Sarina R; Lwi, Sandy J; Casey, James J; Nguyen, Nguyen K; Whalen, Patrick K; Keltner, Dacher; Levenson, Robert W

2015-08-01

The present research examined the effect of the 5-HTTLPR polymorphism in the serotonin transporter gene on objectively coded positive emotional expressions (i.e., laughing and smiling behavior objectively coded using the Facial Action Coding System). Three studies with independent samples of participants were conducted. Study 1 examined young adults watching still cartoons. Study 2 examined young, middle-aged, and older adults watching a thematically ambiguous yet subtly amusing film clip. Study 3 examined middle-aged and older spouses discussing an area of marital conflict (that typically produces both positive and negative emotion). Aggregating data across studies, results showed that the short allele of 5-HTTLPR predicted heightened positive emotional expressions. Results remained stable when controlling for age, gender, ethnicity, and depressive symptoms. These findings are consistent with the notion that the short allele of 5-HTTLPR functions as an emotion amplifier, which may confer heightened susceptibility to environmental conditions. (c) 2015 APA, all rights reserved).

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

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

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

Natural language indicators of differential gene regulation in the human immune system.

PubMed

Mehl, Matthias R; Raison, Charles L; Pace, Thaddeus W W; Arevalo, Jesusa M G; Cole, Steve W

2017-11-21

Adverse social conditions have been linked to a conserved transcriptional response to adversity (CTRA) in circulating leukocytes that may contribute to social gradients in disease. However, the CNS mechanisms involved remain obscure, in part because CTRA gene-expression profiles often track external social-environmental variables more closely than they do self-reported internal affective states such as stress, depression, or anxiety. This study examined the possibility that variations in patterns of natural language use might provide more sensitive indicators of the automatic threat-detection and -response systems that proximally regulate autonomic induction of the CTRA. In 22,627 audio samples of natural speech sampled from the daily interactions of 143 healthy adults, both total language output and patterns of function-word use covaried with CTRA gene expression. These language features predicted CTRA gene expression substantially better than did conventional self-report measures of stress, depression, and anxiety and did so independently of demographic and behavioral factors (age, sex, race, smoking, body mass index) and leukocyte subset distributions. This predictive relationship held when language and gene expression were sampled more than a week apart, suggesting that associations reflect stable individual differences or chronic life circumstances. Given the observed relationship between personal expression and gene expression, patterns of natural language use may provide a useful behavioral indicator of nonconsciously evaluated well-being (implicit safety vs. threat) that is distinct from conscious affective experience and more closely tracks the neurobiological processes involved in peripheral gene regulation. Copyright © 2017 the Author(s). Published by PNAS.

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

PubMed

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

2006-12-01

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

A Systems Biology Analysis Unfolds the Molecular Pathways and Networks of Two Proteobacteria in Spaceflight and Simulated Microgravity Conditions.

PubMed

Roy, Raktim; Shilpa, P Phani; Bagh, Sangram

2016-09-01

Bacteria are important organisms for space missions due to their increased pathogenesis in microgravity that poses risks to the health of astronauts and for projected synthetic biology applications at the space station. We understand little about the effect, at the molecular systems level, of microgravity on bacteria, despite their significant incidence. In this study, we proposed a systems biology pipeline and performed an analysis on published gene expression data sets from multiple seminal studies on Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium under spaceflight and simulated microgravity conditions. By applying gene set enrichment analysis on the global gene expression data, we directly identified a large number of new, statistically significant cellular and metabolic pathways involved in response to microgravity. Alteration of metabolic pathways in microgravity has rarely been reported before, whereas in this analysis metabolic pathways are prevalent. Several of those pathways were found to be common across studies and species, indicating a common cellular response in microgravity. We clustered genes based on their expression patterns using consensus non-negative matrix factorization. The genes from different mathematically stable clusters showed protein-protein association networks with distinct biological functions, suggesting the plausible functional or regulatory network motifs in response to microgravity. The newly identified pathways and networks showed connection with increased survival of pathogens within macrophages, virulence, and antibiotic resistance in microgravity. Our work establishes a systems biology pipeline and provides an integrated insight into the effect of microgravity at the molecular systems level. Systems biology-Microgravity-Pathways and networks-Bacteria. Astrobiology 16, 677-689.

Robustness of linear quadratic state feedback designs in the presence of system uncertainty. [application to Augmentor Wing Jet STOL Research Aircraft flare control autopilot design

NASA Technical Reports Server (NTRS)

Patel, R. V.; Toda, M.; Sridhar, B.

1977-01-01

The paper deals with the problem of expressing the robustness (stability) property of a linear quadratic state feedback (LQSF) design quantitatively in terms of bounds on the perturbations (modeling errors or parameter variations) in the system matrices so that the closed-loop system remains stable. Nonlinear time-varying and linear time-invariant perturbations are considered. The only computation required in obtaining a measure of the robustness of an LQSF design is to determine the eigenvalues of two symmetric matrices determined when solving the algebraic Riccati equation corresponding to the LQSF design problem. Results are applied to a complex dynamic system consisting of the flare control of a STOL aircraft. The design of the flare control is formulated as an LQSF tracking problem.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-07-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

PubMed

Quaedvlieg, N E; Schlaman, H R; Admiraal, P C; Wijting, S E; Stougaard, J; Spaink, H P

1998-11-01

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Proteomic Analysis of Bacillus thuringiensis Strain 4.0718 at Different Growth Phases

PubMed Central

Li, Xiaohui; Ding, Xuezhi; Xia, Liqiu; Sun, Yunjun; Yuan, Can; Yin, Jia

2012-01-01

The growth process of Bacillus thuringiensis Bt4.0718 strain was studied using proteomic technologies. The proteins of Bt whole cells at three phases—middle vegetative, early sporulation, and late sporulation—were extracted with lysis buffer, followed with separation by 2-DE and identified by MALDI-TOF/TOF MS. Bioactive factors such as insecticidal crystal proteins (ICPs) including Cry1Ac(3), Cry2Aa, and BTRX28, immune inhibitor (InhA), and InhA precursor were identified. InhA started to express at the middle vegetative phase, suggesting its contribution to the survival of Bt in the host body. At the early sporulation phase, ICPs started their expression. CotJC, OppA, ORF1, and SpoIVA related to the formation of crystals and spores were identified, the expression characteristics of which ensured the stable formation of crystals and spores. This study provides an important foundation for further exploration of the stable expression of ICPs, the smooth formation of crystals, and the construction of recombinant strains. PMID:22649324

Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC.

PubMed

Yan, Li; Hu, Rui; Tu, Song; Cheng, Wen-Jun; Zheng, Qiong; Wang, Jun-Wen; Kan, Wu-Sheng; Ren, Yi-Jun

2015-01-01

TNFα played a dominant role in the development and progression of rheumatoid arthritis (RA). Clinical trials proved the efficacies of anti-TNFα agents for curing RA. However, most researchers were concentrating on their abilities of neutralizing TNFα, the potencies of different anti-TNFα agents varied a lot due to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). For better understanding and differentiating the potentiality of various candidate anti-TNF reagents at the stage of new drug research and development, present study established a cell model expressing the transmembrane TNFα for usage in in vitro ADCC or CDC assay, meanwhile, the assay protocol described here could provide guidelines for screening macromolecular antibody drugs. A stable cell subline bearing transmembrane TNFα was first established by conventional transfection method, the expression of transmembrane TNFα was approved by flow cytometer, and the performance of the stable subline in ADCC and CDC assay was evaluated, using human peripheral blood mononuclear cells as effector cells, and Adalimumab as the anti-TNFα reagent. The stable cell subline demonstrated high level of surface expression of transmembrane TNFα, and Adalimumab exerted both ADCC and CDC effects on this cell model. In conclusion, the stable cell line we established in present research could be used in ADCC or CDC assay for screening antibody drugs, which would provide in-depth understanding of the potencies of candidate antibody drugs in addition to the traditional TNFα neutralizing assay.

RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

PubMed

Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

2013-08-01

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

PubMed

Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

Oxygen-dependent secretion of a bioactive hepcidin-GFP chimera.

PubMed

Chachami, Georgia; Lyberopoulou, Aggeliki; Kalousi, Alkmini; Paraskeva, Efrosyni; Pantopoulos, Kostas; Simos, George

2013-06-14

Hepcidin, a hepatic hormone, regulates serum iron levels by controlling both intestinal iron absorption and iron release from macrophages. Although transcription of hepcidin is controlled by diverse stimuli, it remains elusive if post-transcriptional steps of its production are also regulated. To address this issue, GFP was fused to the C-terminus of hepcidin and the chimeric hepcidin-GFP protein was expressed in hepatoma Huh7 cells. Expression and secretion of hepcidin-GFP were analyzed by fluorescence microscopy or western blotting and its activity was assessed by in vitro biological assays. Transient over-expression of hepcidin-GFP resulted in production and secretion of premature forms. On the other hand, stable low-level expression led to synthesis and secretion of a properly matured hepcidin-GFP. This form was biologically active since it affected appropriately the levels of IRP2 and ferritin in human THP1 monocytes and targeted ferroportin in mouse J774 macrophages. Treatment of hepcidin-GFP expressing cells with hypoxia (0.1% O2) altered the subcellular distribution of pro-hepcidin-GFP and significantly reduced the secretion of mature hepcidin-GFP. Our hepcidin-GFP expression system allows the investigation of post-transcriptional processing of hepcidin and implicates hypoxia in its secretion control. Copyright © 2013 Elsevier Inc. All rights reserved.

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds.

PubMed

Cunha, Nicolau B; Murad, André M; Ramos, Gustavo L; Maranhão, Andréia Q; Brígido, Marcelo M; Araújo, Ana Cláudia G; Lacorte, Cristiano; Aragão, Francisco J L; Covas, Dimas T; Fontes, Aparecida M; Souza, Gustavo H M F; Vianna, Giovanni R; Rech, Elíbio L

2011-08-01

The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

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

PubMed

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

2017-03-14

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

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

PubMed

Okuda, Masahiro; Taniguchi, Tomokuni; Takamiya, Osamu

2012-09-01

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

Dopaminergic Modulation of Risky Decision-Making

PubMed Central

Simon, Nicholas W.; Montgomery, Karienn S.; Beas, Blanca S.; Mitchell, Marci R.; LaSarge, Candi L.; Mendez, Ian A.; Bañuelos, Cristina; Vokes, Colin M.; Taylor, Aaron B.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

2012-01-01

Many psychiatric disorders are characterized by abnormal risky decision-making and dysregulated dopamine receptor expression. The current study was designed to determine how different dopamine receptor subtypes modulate risk-taking in young adult rats, using a “Risky Decision-making Task” that involves choices between small “safe” rewards and large “risky” rewards accompanied by adverse consequences. Rats showed considerable, stable individual differences in risk preference in the task, which were not related to multiple measures of reward motivation, anxiety, or pain sensitivity. Systemic activation of D2-like receptors robustly attenuated risk-taking, whereas drugs acting on D1-like receptors had no effect. Systemic amphetamine also reduced risk-taking, an effect which was attenuated by D2-like (but not D1-like) receptor blockade. Dopamine receptor mRNA expression was evaluated in a separate cohort of drug-naive rats characterized in the task. D1 mRNA expression in both nucleus accumbens shell and insular cortex was positively associated with risk-taking, while D2 mRNA expression in orbitofrontal and medial prefrontal cortex predicted risk preference in opposing nonlinear patterns. Additionally, lower levels of D2 mRNA in dorsal striatum were associated with greater risk-taking. These data strongly implicate dopamine signaling in prefrontal corticalstriatal circuitry in modulating decision-making processes involving integration of reward information with risks of adverse consequences. PMID:22131407

Stable, high-level expression of a type I antifreeze protein in Escherichia coli.

PubMed

Solomon, R G; Appels, R

1999-06-01

The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.

Paleoenvironmental conditions in the late Paleogene, Sumatra, Indonesia

NASA Astrophysics Data System (ADS)

O'shea, Natalie; Arthur Bettis, E.; Zaim, Yahdi; Rizal, Yan; Aswan, Aswan; Gunnell, Gregg F.; Zonneveld, John-Paul; Ciochon, Russell L.

2015-11-01

A stratified paleosol sequence exposed in an open pit mine in central Sumatra provides a record of the paleoenvironmental conditions in the lower reaches of a large river system in the late Paleogene (latest Eocene or Oligocene). Morphological, geochemical, and stable isotope data suggest that the sequence represents a mosaic of local environmental conditions changing from estuarine to riverine up section. Weakly expressed soils formed on low-lying estuary surfaces, while more well expressed soils formed on higher, better drained surfaces. Peatlands (coal) with clayey subsoils were along the estuary margins. Well-expressed soils with evidence of clay translocation and chemical weathering become more common higher in the section where alluvial deposits associated with a meandering river are dominant. Stable carbon isotope ratios support a paleolandscape dominated by C3 plants with input by C4 vegetation limited to a few intervals. Finally, whole-rock geochemistry suggests moderate chemical weathering consistent with a tropical locality. This multi-proxy paleoenvironmental reconstruction suggests a highly productive lowland forest environment at this locality in the late Paleogene and provides the first direct examination of the terrestrial environment in Sumatra at this time. The limited fossil record in Island Southeast Asia during this time period is likely a result of poor bone and shell preservation in tropical forest environments combined with a general lack of systematic prospecting. However, our continuing work in this area has produced a relatively diverse assemblage of fossil vertebrates, now including fishes, amphibians, turtles, crocodiles, and mammals, as well as a growing diversity of fossil plants.

Generation of a new Gateway-compatible inducible lentiviral vector platform allowing easy derivation of co-transduced cells.

PubMed

De Groote, Philippe; Grootjans, Sasker; Lippens, Saskia; Eichperger, Chantal; Leurs, Kirsten; Kahr, Irene; Tanghe, Giel; Bruggeman, Inge; De Schamphelaire, Wouter; Urwyler, Corinne; Vandenabeele, Peter; Haustraete, Jurgen; Declercq, Wim

2016-01-01

In contrast to most common gene delivery techniques, lentiviral vectors allow targeting of almost any mammalian cell type, even non-dividing cells, and they stably integrate in the genome. Therefore, these vectors are a very powerful tool for biomedical research. Here we report the generation of a versatile new set of 22 lentiviral vectors with broad applicability in multiple research areas. In contrast to previous systems, our platform provides a choice between constitutive and/or conditional expression and six different C-terminal fusions. Furthermore, two compatible selection markers enable the easy derivation of stable cell lines co-expressing differently tagged transgenes in a constitutive or inducible manner. We show that all of the vector features are functional and that they contribute to transgene overexpression in proof-of-principle experiments.

[Transformation of Chlamydomonas reinhardtii CW-15 with the hygromycin phosphotransferase gene as a selective marker].

PubMed

Ladygin, V G; Butanaev, A M

2002-09-01

To transform Chlamydomonas reinhardtii Dang. Cells, plasmid pCTVHyg was constructed with the use of the Escherichia coli hygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10(3) hygromycin-resistant (HygR) clones per 10(6) recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hpt gene and Ch. reinhardtii nuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.

Minimalist Antibodies and Mimetics: An Update and Recent Applications.

PubMed

Bruce, Virginia J; Ta, Angeline N; McNaughton, Brian R

2016-10-17

The immune system utilizes antibodies to recognize foreign or disease-relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small-molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post-translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli.

PubMed

Songsiriritthigul, Chomphunuch; Buranabanyat, Bancha; Haltrich, Dietmar; Yamabhai, Montarop

2010-04-11

Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose. Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

A Manual for a Laboratory Information Management System (LIMS) For Light Stable Isotopes - Version 7.0

DTIC Science & Technology

1998-01-01

A MANUAL FOR A LABORATORY INFORMATION MANAGEMENT SYSTEM (LIMS) FOR LIGHT STABLE ISOTOPES— VERSION 7.0 U.S. GEOLOGICAL SURVEY Open-File Report 98-284...Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 A MANUAL FOR A LABORATORY INFORMATION MANAGEMENT SYSTEM (LIMS) FOR LIGHT STABLE...Europa Scientific ..................................................120 1 A MANUAL FOR A LABORATORY INFORMATION MANAGEMENT SYSTEM (LIMS) FOR LIGHT STABLE

Tomato Spotted Wilt Virus NSs Protein Supports Infection and Systemic Movement of a Potyvirus and Is a Symptom Determinant

PubMed Central

Gabriel Peralta, Sergio M.; Harte-Maxwell, Patricia A.

2018-01-01

Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro). When expressed in cis, NSs restored pathogenicity and promoted systemic infection of suppressor-deficient TuMV-AS9-GFP in Nicotiana benthamiana and Arabidopsis thaliana. Inactivating mutations were introduced in NSs RNA-binding domain one. A genetic analysis with active and suppressor-deficient NSs, in combination with wild-type and mutant plants lacking essential components of the RNA silencing machinery, showed that the NSs insert is stable when expressed from a potyvirus. NSs can functionally replace potyviral HC-Pro, condition virus susceptibility, and promote systemic infection and symptom development by suppressing antiviral RNA silencing through a mechanism that partially overlaps that of potyviral HC-Pro. The results presented provide new insight into the mechanism of silencing suppression by NSs and its effect on virus infection. PMID:29538326

Tomato Spotted Wilt Virus NSs Protein Supports Infection and Systemic Movement of a Potyvirus and Is a Symptom Determinant.

PubMed

Garcia-Ruiz, Hernan; Gabriel Peralta, Sergio M; Harte-Maxwell, Patricia A

2018-03-14

Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro). When expressed in cis, NSs restored pathogenicity and promoted systemic infection of suppressor-deficient TuMV-AS9-GFP in Nicotiana benthamiana and Arabidopsis thaliana . Inactivating mutations were introduced in NSs RNA-binding domain one. A genetic analysis with active and suppressor-deficient NSs, in combination with wild-type and mutant plants lacking essential components of the RNA silencing machinery, showed that the NSs insert is stable when expressed from a potyvirus. NSs can functionally replace potyviral HC-Pro, condition virus susceptibility, and promote systemic infection and symptom development by suppressing antiviral RNA silencing through a mechanism that partially overlaps that of potyviral HC-Pro. The results presented provide new insight into the mechanism of silencing suppression by NSs and its effect on virus infection.

Drosophila segmentation: supercomputer simulation of prepattern hierarchy.

PubMed

Hunding, A; Kauffman, S A; Goodwin, B C

1990-08-09

Spontaneous prepattern formation in a two level hierarchy of reaction-diffusion systems is simulated in three space co-ordinates and time, mimicking gap gene and primary pair-rule gene expression. The model rests on the idea of Turing systems of the second kind, in which one prepattern generates position dependent rate constants for a subsequent reaction-diffusion system. Maternal genes are assumed responsible for setting up gradients from the anterior and posterior ends, one of which is needed to stabilize a double period prepattern suggested to underly the read out of the gap genes. The resulting double period pattern in turn stabilizes the next prepattern in the hierarchy, which has a short wavelength with many characteristics of the stripes seen in actual primary pair-rule gene expression. Without such hierarchical stabilization, reaction-diffusion mechanisms yield highly patchy short wave length patterns, and thus unreliable stripes. The model yields seven stable stripes located in the middle of the embryo, with the potential for additional expression near the poles, as observed experimentally. The model does not rely on specific chemical reaction kinetics, rather the effect is general to many such kinetic schemes. This makes it robust to parameter changes, and it has good potential for adapting to size and shape changes as well. The study thus suggests that the crucial organizing principle in early Drosophila embryogenesis is based on global field mechanisms, not on particular local interactions.

Mycophenolate Mofetil Treatment of Systemic Sclerosis Reduces Myeloid Cell Numbers and Attenuates the Inflammatory Gene Signature in Skin.

PubMed

Hinchcliff, Monique; Toledo, Diana M; Taroni, Jaclyn N; Wood, Tammara A; Franks, Jennifer M; Ball, Michael S; Hoffmann, Aileen; Amin, Sapna M; Tan, Ainah U; Tom, Kevin; Nesbeth, Yolanda; Lee, Jungwha; Ma, Madeleine; Aren, Kathleen; Carns, Mary A; Pioli, Patricia A; Whitfield, Michael L

2018-01-31

Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2011-01-01

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

Stability of Reference Gene Expression After Porcine Sapelovirus Infection in Porcine Intestinal Epithelial Cells.

PubMed

Huang, Yong; Chen, Yabing; Sun, Huan; Lan, Daoliang

2016-01-01

Intestinal epithelial cells, which serve as the first physical barrier to protect intestinal tract from external antigens, have an important role in the local innate immunity. Screening of reference genes that have stable expression levels after viral infection in porcine intestinal epithelial cells is critical for ensuring the reliability of the expression analysis on anti-infection genes in porcine intestinal epithelial cells. In this study, nine common reference genes in pigs, including ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, TBP, YWHAZ, and RPL32, were chosen as the candidate reference genes. Porcine sapelovirus (PSV) was used as a model virus to infect porcine intestinal epithelial cell line (IPEC-J2). The expression stability of the nine genes was assessed by the geNorm, NormFinder, and BestKeeper software. Moreover, RefFinder program was used to evaluate the analytical results of above three softwares, and a relative expression experiment of selected target gene was used to verify the analysis results. The comprehensive results indicated that the gene combination of TBP and RPL32 has the most stable expression, which could be considered as an appropriate reference gene for research on gene expression after PSV infection in IPEC-J2cells. The results provided essential data for expression analysis of anti-infection genes in porcine intestinal epithelial cells.

Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces.

PubMed

Sevillano, Laura; Díaz, Margarita; Santamaría, Ramón I

2017-09-26

The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. Graphical abstract Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.

Systems Level Analysis of Systemic Sclerosis Shows a Network of Immune and Profibrotic Pathways Connected with Genetic Polymorphisms

PubMed Central

Mahoney, J. Matthew; Taroni, Jaclyn; Martyanov, Viktor; Wood, Tammara A.; Greene, Casey S.; Pioli, Patricia A.; Hinchcliff, Monique E.; Whitfield, Michael L.

2015-01-01

Systemic sclerosis (SSc) is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited) are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6–12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA) genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected and related to a patients underlying genetic risk. PMID:25569146

Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

PubMed

Nair, Nisha R; Chidambareswaren, M; Manjula, S

2014-09-01

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

BOREAS TE-5 CO2 Concentration and Stable Isotope Composition

NASA Technical Reports Server (NTRS)

Hall, Forrest G. (Editor); Curd, Shelaine (Editor); Ehleriinger, Jim; Brooks, J. Renee; Flanagan, Larry

2000-01-01

The BOREAS TE-5 team collected measurements in the NSA and SSA on gas exchange, gas composition, and tree growth. This data set contains measurements of the concentration and stable carbon (C-13/C-12 and oxygen (O-18/O-16) isotope ratios of atmospheric CO2 in air samples collected at different heights within forest canopies. The data were collected to determine the influence of photosynthesis and respiration by the forest ecosystems on the concentration and stable isotope ratio of atmospheric CO2 These measurements were collected at the SSA during each 1994 IFC at OJP, OBS, and OA sites. Measurements were also collected at the NSA during each 1994 IFC at the OJP, T6R5S TE UBS, and T2Q6A TE OA sites. The stable isotope ratios are expressed using standard delta notation and in units of per mil. The isotope ratios are expressed relative to the international standard, PDB, for both carbon and oxygen samples. The data are stored in tabular ASCII files. The data files are available on a CD-ROM (see document number 20010000884), or from the Oak Ridge National Laboratory (ORNL) Distributed Activity Archive Center (DAAC).

Toxin–antitoxin systems

PubMed Central

Unterholzner, Simon J; Poppenberger, Brigitte; Rozhon, Wilfried

2013-01-01

Toxin–antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin. The toxins of all known TA systems are proteins while the antitoxins are either proteins or non-coding RNAs. Based on the molecular nature of the antitoxin and its mode of interaction with the toxin the TA modules are currently grouped into five classes. In general, the toxin is more stable than the antitoxin but the latter is expressed to a higher level. If supply of the antitoxin stops, for instance under special growth conditions or by plasmid loss in case of plasmid encoded TA systems, the antitoxin is rapidly degraded and can no longer counteract the toxin. Consequently, the toxin becomes activated and can act on its cellular targets. Typically, TA toxins act on crucial cellular processes including translation, replication, cytoskeleton formation, membrane integrity, and cell wall biosynthesis. TA systems and their components are also versatile tools for a multitude of purposes in basic research and biotechnology. Currently, TA systems are frequently used for selection in cloning and for single protein expression in living bacterial cells. Since several TA toxins exhibit activity in yeast and mammalian cells they may be useful for applications in eukaryotic systems. TA modules are also considered as promising targets for the development of antibacterial drugs and their potential to combat viral infection may aid in controlling infectious diseases. PMID:24251069

Coupling of damped and growing modes in unstable shear flow

DOE PAGES

Fraser, A. E.; Terry, P. W.; Zweibel, E. G.; ...

2017-06-14

Analysis of the saturation of the Kelvin-Helmholtz instability is undertaken to determine the extent to which the conjugate linearly stable mode plays a role. For a piecewise-continuous mean flow profile with constant shear in a fixed layer, it is shown that the stable mode is nonlinearly excited, providing an injection-scale sink of the fluctuation energy similar to what has been found for gyroradius-scale drift-wave turbulence. Quantitative evaluation of the contribution of the stable mode to the energy balance at the onset of saturation shows that nonlinear energy transfer to the stable mode is as significant as energy transfer to smallmore » scales in balancing energy injected into the spectrum by the instability. The effect of the stable mode on momentum transport is quantified by expressing the Reynolds stress in terms of stable and unstable mode amplitudes at saturation, from which it is found that the stable mode can produce a sizable reduction in the momentum flux.« less

Coupling of damped and growing modes in unstable shear flow

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

Fraser, A. E.; Terry, P. W.; Zweibel, E. G.

Analysis of the saturation of the Kelvin-Helmholtz instability is undertaken to determine the extent to which the conjugate linearly stable mode plays a role. For a piecewise-continuous mean flow profile with constant shear in a fixed layer, it is shown that the stable mode is nonlinearly excited, providing an injection-scale sink of the fluctuation energy similar to what has been found for gyroradius-scale drift-wave turbulence. Quantitative evaluation of the contribution of the stable mode to the energy balance at the onset of saturation shows that nonlinear energy transfer to the stable mode is as significant as energy transfer to smallmore » scales in balancing energy injected into the spectrum by the instability. The effect of the stable mode on momentum transport is quantified by expressing the Reynolds stress in terms of stable and unstable mode amplitudes at saturation, from which it is found that the stable mode can produce a sizable reduction in the momentum flux.« less

Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

PubMed Central

Lavitrano, Marialuisa; Bacci, Maria Laura; Forni, Monica; Lazzereschi, Davide; Di Stefano, Carla; Fioretti, Daniela; Giancotti, Paola; Marfé, Gabriella; Pucci, Loredana; Renzi, Luigina; Wang, Hongjun; Stoppacciaro, Antonella; Stassi, Giorgio; Sargiacomo, Massimo; Sinibaldi, Paola; Turchi, Valeria; Giovannoni, Roberto; Della Casa, Giacinto; Seren, Eraldo; Rossi, Giancarlo

2002-01-01

A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models. PMID:12393815

Morphogenesis in Plants: Modeling the Shoot Apical Meristem, and Possible Applications

NASA Technical Reports Server (NTRS)

Mjolsness, Eric; Gor, Victoria; Meyerowitz, Elliot; Mann, Tobias

1998-01-01

A key determinant of overall morphogenesis in flowering plants such as Arabidopsis thaliana is the shoot apical meristem (growing tip of a shoot). Gene regulation networks can be used to model this system. We exhibit a very preliminary two-dimensional model including gene regulation and intercellular signaling, but omitting cell division and dynamical geometry. The model can be trained to have three stable regions of gene expression corresponding to the central zone, peripheral zone, and rib meristem. We also discuss a space-engineering motivation for studying and controlling the morphogenesis of plants using such computational models.

Self-dual monopoles and toda molecules

NASA Astrophysics Data System (ADS)

Ganoulis, N.; Goddard, P.; Olive, D.

1982-07-01

Stable static solutions to a gauge field theory with a Higgs field in the adjoint representation and with vanishing self-coupling are self-dual in the sense of Bogomolny. Leznov and Saveliev showed that a specific form of spherical symmetry reduces these equations to a modified form of the Toda molecule equations associated with the overall gauge symmetry G. Values of the constants of integration are found in terms of the distant Higgs field, guaranteeing regularity of the solution at the origin. The expressions hold for any simple Lie group G, depending on G via its root system.

Advanced Thermally Stable Jet Fuels Development Program Annual Report. Volume 1. Model and Experiment System Development

DTIC Science & Technology

1990-09-01

like to acknowledge R. J. Kottenstette, H. P. Stephens, D . M. Haaland, A . S. Geller, W. M. Trott, S. J. Martin, A . J. Lammie, B. J. Lammie, D . S. Blair...be expressed as F - D q?, where D is the diffusion coefficient, and q the scattering wave vector q- 4wnsin(8/2)/ A i, with n the liquid refractive...tubes C and D were generated using 11.4 mg/L JFA-5 added to the JP-8 fuel. The IR spectrum from tube A contained primarily a carbonate deposit with a

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in Buckwheat (Fagopyrum esculentum) Based on Transcriptome Sequence Data

PubMed Central

Demidenko, Natalia V.; Logacheva, Maria D.; Penin, Aleksey A.

2011-01-01

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications – geNorm, NormFinder and BestKeeper - were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species. PMID:21589908

The physical characteristics of human proteins in different biological functions.

PubMed

Wang, Tengjiao; Tang, Hailin

2017-01-01

The physical properties of gene products are the foundation of their biological functions. In this study, we systematically explored relationships between physical properties and biological functions. The physical properties including origin time, evolution pressure, mRNA and protein stability, molecular weight, hydrophobicity, acidity/alkaline, amino acid compositions, and chromosome location. The biological functions are defined from 4 aspects: biological process, molecular function, cellular component and cell/tissue/organ expression. We found that the proteins associated with basic material and energy metabolism process originated earlier, while the proteins associated with immune, neurological system process etc. originated later. Tissues may have a strong influence on evolution pressure. The proteins associated with energy metabolism are double-stable. Immune and peripheral cell proteins tend to be mRNA stable/protein unstable. There are very few function items with double-unstable of mRNA and protein. The proteins involved in the cell adhesion tend to consist of large proteins with high proportion of small amino acids. The proteins of organic acid transport, neurological system process and amine transport have significantly high hydrophobicity. Interestingly, the proteins involved in olfactory receptor activity tend to have high frequency of aromatic, sulfuric and hydroxyl amino acids.

The physical characteristics of human proteins in different biological functions

PubMed Central

Tang, Hailin

2017-01-01

The physical properties of gene products are the foundation of their biological functions. In this study, we systematically explored relationships between physical properties and biological functions. The physical properties including origin time, evolution pressure, mRNA and protein stability, molecular weight, hydrophobicity, acidity/alkaline, amino acid compositions, and chromosome location. The biological functions are defined from 4 aspects: biological process, molecular function, cellular component and cell/tissue/organ expression. We found that the proteins associated with basic material and energy metabolism process originated earlier, while the proteins associated with immune, neurological system process etc. originated later. Tissues may have a strong influence on evolution pressure. The proteins associated with energy metabolism are double-stable. Immune and peripheral cell proteins tend to be mRNA stable/protein unstable. There are very few function items with double-unstable of mRNA and protein. The proteins involved in the cell adhesion tend to consist of large proteins with high proportion of small amino acids. The proteins of organic acid transport, neurological system process and amine transport have significantly high hydrophobicity. Interestingly, the proteins involved in olfactory receptor activity tend to have high frequency of aromatic, sulfuric and hydroxyl amino acids. PMID:28459865

Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma

PubMed Central

Peciak, Joanna; Stec, Wojciech J; Treda, Cezary; Ksiazkiewicz, Magdalena; Janik, Karolina; Popeda, Marta; Smolarz, Maciej; Rosiak, Kamila; Hulas-Bigoszewska, Krystyna; Och, Waldemar; Rieske, Piotr; Stoczynska-Fidelus, Ewelina

2017-01-01

Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene. PMID:28123609

Parameters optimization for magnetic resonance coupling wireless power transmission.

PubMed

Li, Changsheng; Zhang, He; Jiang, Xiaohua

2014-01-01

Taking maximum power transmission and power stable transmission as research objectives, optimal design for the wireless power transmission system based on magnetic resonance coupling is carried out in this paper. Firstly, based on the mutual coupling model, mathematical expressions of optimal coupling coefficients for the maximum power transmission target are deduced. Whereafter, methods of enhancing power transmission stability based on parameters optimal design are investigated. It is found that the sensitivity of the load power to the transmission parameters can be reduced and the power transmission stability can be enhanced by improving the system resonance frequency or coupling coefficient between the driving/pick-up coil and the transmission/receiving coil. Experiment results are well conformed to the theoretical analysis conclusions.

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

PubMed Central

Park, Jong-Won; Beyene, Getu; Buenrostro-Nava, Marco T.; Molina, Joe; Wang, Xiaofeng; Ciomperlik, Jessica J.; Manabayeva, Shuga A.; Alvarado, Veria Y.; Rathore, Keerti S.; Scholthof, Herman B.; Mirkov, T. Erik

2013-01-01

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. PMID:23799071

Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

PubMed Central

Yang, Wen-Kai; Kang, Chao-Kai; Chang, Chia-Hao; Hsu, An-Di; Lee, Tsung-Han; Hwang, Pung-Pung

2013-01-01

FXYD proteins are novel regulators of Na+-K+-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies. PMID:23383199

Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations.

PubMed

Jiang, WenZhi; Yang, Bing; Weeks, Donald P

2014-01-01

The newly developed CRISPR/Cas9 system for targeted gene knockout or editing has recently been shown to function in plants in both transient expression systems as well as in primary T1 transgenic plants. However, stable transmission of genes modified by the Cas9/single guide RNA (sgRNA) system to the T2 generation and beyond has not been demonstrated. Here we provide extensive data demonstrating the efficiency of Cas9/sgRNA in causing modification of a chromosomally integrated target reporter gene during early development of transgenic Arabidopsis plants and inheritance of the modified gene in T2 and T3 progeny. Efficient conversion of a nonfunctional, out-of-frame GFP gene to a functional GFP gene was confirmed in T1 plants by the observation of green fluorescent signals in leaf tissues as well as the presence of mutagenized DNA sequences at the sgRNA target site within the GFP gene. All GFP-positive T1 transgenic plants and nearly all GFP-negative plants examined contained mutagenized GFP genes. Analyses of 42 individual T2 generation plants derived from 6 different T1 progenitor plants showed that 50% of T2 plants inherited a single T-DNA insert. The efficiency of the Cas9/sgRNA system and stable inheritance of edited genes point to the promise of this system for facile editing of plant genes.

EBV and systemic lupus erythematosus: a new perspective.

PubMed

Gross, Andrew J; Hochberg, Donna; Rand, William M; Thorley-Lawson, David A

2005-06-01

We have proposed that EBV uses mature B cell biology to access memory B cells as a site of persistent infection. A central feature of this model is that EBV adapts its gene expression profile to the state of the B cell it resides in and that the level of infection is stable over time. This led us to question whether changes in the behavior or regulation of mature B cells would alter the state of EBV persistence. To investigate this, we studied the impact of systemic lupus erythematosus (SLE), a disease characterized by immune dysfunction, on EBV infection. We show that patients with SLE have abnormally high frequencies of EBV-infected cells in their blood, and this is associated with the occurrence of SLE disease flares. Although patients with SLE have frequencies of infected cells comparable to those seen in immunosuppressed patients, in SLE the effect was independent of immunosuppressive therapy. Aberrant expression of viral lytic (BZLF1) and latency (latency membrane proteins 1 and 2a) genes was also detected in the blood of SLE patients. We conclude that the abnormal regulation of EBV infection in SLE patients reflects the sensitivity of the virus to perturbation of the immune system.

Selection of reference genes for tissue/organ samples on day 3 fifth-instar larvae in silkworm, Bombyx mori.

PubMed

Wang, Genhong; Chen, Yanfei; Zhang, Xiaoying; Bai, Bingchuan; Yan, Hao; Qin, Daoyuan; Xia, Qingyou

2018-06-01

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth-instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori. © 2018 Wiley Periodicals, Inc.

Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells.

PubMed

Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

2016-01-01

Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future.

Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells

PubMed Central

Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

2016-01-01

Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future. PMID:27362942

Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

PubMed Central

Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

2016-01-01

Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

Interfacial polymerization for colorimetric labeling of protein expression in cells.

PubMed

Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

2014-01-01

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973.

PubMed

Wendt, Kristen E; Ungerer, Justin; Cobb, Ryan E; Zhao, Huimin; Pakrasi, Himadri B

2016-06-23

As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.

CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973

DOE PAGES

Wendt, Kristen E.; Ungerer, Justin; Cobb, Ryan E.; ...

2016-06-23

As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructsmore » containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. In conclusion, high expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.« less

CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973

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

Wendt, Kristen E.; Ungerer, Justin; Cobb, Ryan E.

As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructsmore » containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. In conclusion, high expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.« less

Peripheral Endocannabinoid System Activity in Patients Treated With Sibutramine

PubMed Central

Engeli, Stefan; Heusser, Karsten; Janke, Jürgen; Gorzelniak, Kerstin; Bátkai, Sándor; Pacher, Pál; Harvey-White, Judith; Luft, Friedrich C.; Jordan, Jens

2008-01-01

Objective The endocannabinoid system (ECS) promotes weight gain and obesity-associated metabolic changes. Weight loss interventions may influence obesity-associated risk indirectly through modulation of the peripheral ECS. We investigated the effect of acute and chronic treatment with sibutramine on components of the peripheral ECS. Methods and Procedures Twenty obese otherwise healthy patients received randomized, double-blind, crossover treatment with placebo and 15 mg/day sibutramine for 5 days each, followed by 12 weeks open-label sibutramine treatment. We determined circulating anandamide and 2-arachidonoylglycerol and expression levels of endocannabinoid genes in subcutaneous abdominal adipose tissue biopsies. Results Body weight was stable during the acute treatment period and decreased by 6.0 ± 0.8 kg in those patients completing 3 months of sibutramine treatment (P < 0.05). Circulating endocannabinoids and the expression of ECS genes did not change with acute or chronic sibutramine treatment. Discussion The ECS is activated in obesity. We did not find any influence of 5% body weight loss induced by sibutramine on circulating levels of endocannabinoids and adipose-tissue expression of endocannabinoid genes in obese subjects. These data confirm our previous findings on dietary weight loss and suggest that the dysregulation of the ECS may be a cause rather than a consequence of obesity. PMID:18356837

Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L.

PubMed

Moreira, Viviane S; Soares, Virgínia L F; Silva, Raner J S; Sousa, Aurizangela O; Otoni, Wagner C; Costa, Marcio G C

2018-05-01

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana , coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA , for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.

Tissue-associated self-antigens containing exosomes: Role in allograft rejection.

PubMed

Sharma, Monal; Ravichandran, Ranjithkumar; Bansal, Sandhya; Bremner, Ross M; Smith, Michael A; Mohanakumar, T

2018-06-15

Exosomes are extracellular vesicles that express self-antigens (SAgs) and donor human leukocyte antigens. Tissue-specific exosomes can be detected in the circulation following lung, heart, kidney and islet cell transplantations. We collected serum samples from patients who had undergone lung (n = 30), heart (n = 8), or kidney (n = 15) transplantations to isolate circulating exosomes. Exosome purity was analyzed by Western blot, using CD9 exosome-specific markers. Tissue-associated lung SAgs, collagen V (Col-V) and K-alpha 1 tubulin (Kα1T), heart SAgs, myosin and vimentin, and kidney SAgs, fibronectin and collagen IV (Col-IV), were identified using western blot. Lung transplant recipients diagnosed with bronchiolitis obliterans syndrome had exosomes with higher expression of Col-V (4.2-fold) and Kα1T (37.1-fold) than stable. Exosomes isolated from heart transplant recipients diagnosed with coronary artery vasculopathy had a 3.9-fold increase in myosin and a 4.7-fold increase in vimentin compared with stable. Further, Kidney transplant recipients diagnosed with transplant glomerulopathy had circulating exosomes with a 2-fold increased expression of fibronectin and 2.5-fold increase in Col-IV compared with stable. We conclude that circulating exosomes with tissue associated SAgs have the potential to be a noninvasive biomarker for allograft rejection. Copyright © 2018. Published by Elsevier Inc.

Application of CRISPR/Cas9 system in breeding of new antiviral plant germplasm.

PubMed

Zhang, Dao-wei; Zhang, Chao-fan; Dong, Fang; Huang, Yan-lan; Zhang, Ya; Zhou, Hong

2016-09-01

With the development and improvement of CRISPR/Cas9 system in genomic editing technology, the system has been applied to the prevention and control of animal viral infectious diseases, which has made considerable achievements. It has also been applied to the study of highly efficient gene targeting editing in plant virus genomes. The CRISPR/Cas9-mediated targeted gene modification has not only achieved the genome editing of plant DNA virus, but also showed the genome editing potential of plant RNA virus. In addition, the CRISPR/Cas9 system functions at the gene transcriptional and post-transcriptional level, indicating that the system could regulate the replication of plant viruses through different ways. Compared with other plant viral disease control strategies, this system is more accurate in genome editing, more stable in gene expression regulation, and has broader spectrum of resistance to virus disease. In this review, we summarized the advantages, main problems and development tendency of CRISPR/cas9 system in breeding of new antiviral plant germplasms.

Kinetic Equation for an Unstable Plasma

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

Balescu, R.

1963-01-01

A kinetic equation is derived for the description of the evolution in time of the distribution of velocities in a spatially homogeneous ionized gas that, at the initial time, is able to sustain exponentially growing oscillations. This equation is expressed in terms of a functional of the distribution finction that obeys the same integral equation as in the stable case. Although the method of solution used in the stable case breaks down, the equation can still be solved in closed form under unstable conditions, and hence an explicit form of the kinetic equation is obtained. The latter contains the normalmore » collision term and a new additional term describing the stabilization of the plasma. The latter acts through friction and diffusion and brings the plasma into a state of neutral stability. From there on the system evolves toward thermal equilibrium under the action of the normal collision term as well as of an additional Fokker-Planck- like term with timedependent coefficients, which however becomes less and less efficient as the plasma approaches equilibrium.« less

Epithelioid hemangioendotheliomas of the liver and lung in children and adolescents.

PubMed

Hettmer, Simone; Andrieux, Geoffroy; Hochrein, Jochen; Kurz, Philipp; Rössler, Jochen; Lassmann, Silke; Werner, Martin; von Bubnoff, Nikolas; Peters, Christoph; Koscielniak, Ewa; Sparber-Sauer, Monika; Niemeyer, Charlotte; Mentzel, Thomas; Busch, Hauke; Boerries, Melanie

2017-12-01

Epithelioid hemangioendothelioma (EHE) is a rare, vascular sarcoma. Visceral forms arise in the liver/ lungs. We review the clinical and molecular phenotype of pediatric visceral EHE based on the case of a 9-year-old male child with EHE of the liver/lungs. His tumor expressed the EHE-specific fusion oncogene WWTR1-CAMTA1. Molecular characterization revealed a low somatic mutation rate and activated interferon signaling, angiogenesis regulation, and blood vessel remodeling. After polychemotherapy and resection of lung tumors, residual disease remained stable on oral lenalidomide. Literature review identified another 24 children with EHE of the liver/lungs. Most presented with multifocal, systemic disease. Only those who underwent complete resection achieved complete remission. Four children experienced rapid progression and died. In six children, disease remained stable for years without therapy. Two patients died from progressive EHE 21 and 24 years after first diagnosis. Natural evolution of pediatric visceral EHE is variable, and long-term prognosis remains unclear. © 2017 Wiley Periodicals, Inc.

Oil Palm Defensin: A Thermal Stable Peptide that Restricts the Mycelial Growth of Ganoderma boninense.

PubMed

Tan, Yung-Chie; Ang, Cheng-Liang; Wong, Mui-Yun; Ho, Chai-Ling

2016-01-01

Plant defensins are plant defence peptides that have many different biological activities, including antifungal, antimicrobial, and insecticidal activities. A cDNA (EgDFS) encoding defensin was isolated from Elaeis guineensis. The open reading frame of EgDFS contained 231 nucleotides encoding a 71-amino acid protein with a predicted molecular weight at 8.69 kDa, and a potential signal peptide. The eight highly conserved cysteine sites in plant defensins were also conserved in EgDFS. The EgDFS sequence lacking 30 amino acid residues at its N-terminus (EgDFSm) was cloned into Escherichia coli BL21 (DE3) pLysS and successfully expressed as a soluble recombinant protein. The recombinant EgDFSm was found to be a thermal stable peptide which demonstrated inhibitory activity against the growth of G. boninense possibly by inhibiting starch assimilation. The role of EgDFSm in oil palm defence system against the infection of pathogen G. boninense was discussed.

Automated structure determination of proteins with the SAIL-FLYA NMR method.

PubMed

Takeda, Mitsuhiro; Ikeya, Teppei; Güntert, Peter; Kainosho, Masatsune

2007-01-01

The labeling of proteins with stable isotopes enhances the NMR method for the determination of 3D protein structures in solution. Stereo-array isotope labeling (SAIL) provides an optimal stereospecific and regiospecific pattern of stable isotopes that yields sharpened lines, spectral simplification without loss of information, and the ability to collect rapidly and evaluate fully automatically the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as those that can be analyzed using conventional methods. Here, we describe a protocol for the preparation of SAIL proteins by cell-free methods, including the preparation of S30 extract and their automated structure analysis using the FLYA algorithm and the program CYANA. Once efficient cell-free expression of the unlabeled or uniformly labeled target protein has been achieved, the NMR sample preparation of a SAIL protein can be accomplished in 3 d. A fully automated FLYA structure calculation can be completed in 1 d on a powerful computer system.

GPS Constraints on the Spatial Distribution of Extension in the Ethiopian Highlands and Main Ethiopian Rift

NASA Astrophysics Data System (ADS)

Amere, Y. B.; Bendick, R. O.; Fisseha, S.; Lewi, E.; Reilinger, R. E.; King, R. W.; Kianji, G.

2014-12-01

27 campaign and 17 continuous GPS sites spanning the Ethiopian Highlands, Main Ethiopian Rift (MER), and Somali Platform in Ethiopia and Eritrea were measured for varying durations between 1995 and 2014. Velocities at these sites show that present day strain in NE Africa is not localized only in the Afar depression and MER system. Rather, velocities as high as 6 mm/yr relative to stable Nubia occur in the central Ethiopian highlands west of the rift bounding faults; the northern and southern Ethiopian highlands host velocities as high as 3 mm/yr. These approach the magnitude of Nubia-Somalia spreading accommodated within the rift itself of 6 + 1 mm/yr with an azimuth of N770E. The combination of distributed low strain rate deformation contiguous with higher strain rate plate boundary deformation is similar to that expressed in other tectonically active continental settings like Basin and Range and Tibetan Plateau.Keywords: deformation, localized, distributed, strain, stable Nubia.

Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures.

PubMed

Kong, D; Gentz, R; Zhang, J

1998-03-01

Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2-6 × 10(6) cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4-10 mg/L. Specific productivity was maintained at 1.8-3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted.

Clutch pressure estimation for a power-split hybrid transmission using nonlinear robust observer

NASA Astrophysics Data System (ADS)

Zhou, Bin; Zhang, Jianwu; Gao, Ji; Yu, Haisheng; Liu, Dong

2018-06-01

For a power-split hybrid transmission, using the brake clutch to realize the transition from electric drive mode to hybrid drive mode is an available strategy. Since the pressure information of the brake clutch is essential for the mode transition control, this research designs a nonlinear robust reduced-order observer to estimate the brake clutch pressure. Model uncertainties or disturbances are considered as additional inputs, thus the observer is designed in order that the error dynamics is input-to-state stable. The nonlinear characteristics of the system are expressed as the lookup tables in the observer. Moreover, the gain matrix of the observer is solved by two optimization procedures under the constraints of the linear matrix inequalities. The proposed observer is validated by offline simulation and online test, the results have shown that the observer achieves significant performance during the mode transition, as the estimation error is within a reasonable range, more importantly, it is asymptotically stable.

D/H ratios and hydrogen exchangeability of type-II kerogens with increasing thermal maturity

USGS Publications Warehouse

Lis, G.P.; Schimmelmann, A.; Mastalerz, Maria

2006-01-01

Stable isotope ratios of non-exchangeable hydrogen (??Dn) and of carbon were measured in type-II kerogens from two suites of Late Devonian to Early Mississippian black shale, one from the New Albany Shale (Illinois Basin) and the other from the Exshaw Formation (Alberta Basin). The largely marine-derived organic matter had similar original stable isotope ratios, but today the suites of kerogens express gradients in thermal maturity that have altered their chemical and isotopic compositions. In both suites, ??D n values increase with maturation up to a vitrinite reflectance of Ro 1.5%, then level out. Increasing ??Dn values suggest isotopic exchange of organic hydrogen with water-derived deuterium and/or preferential loss of 1H-enriched chemical moieties from kerogen during maturation. The resulting changes in ??Dn values are altering the original hydrogen isotopic paleoenvironmental signal in kerogen, albeit in a systematic fashion. The specific D/H response of each kerogen suite through maturation correlates with H/C elemental ratio and can therefore be corrected to yield paleoenvironmentally relevant information for a calibrated system. With increasing thermal maturity, the abundance of hydrogen in the kerogen that is isotopically exchangeable with water hydrogen (expressed as Hex, in % of total hydrogen) first decreases to reach a minimum at Ro ??? 0.8-1.1%, followed by a substantial increase at higher thermal maturity. ?? 2005 Elsevier Ltd. All rights reserved.

Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

PubMed

Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

2015-01-01

Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

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

Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir; Ahmadvand, Davoud

2011-11-01

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for highmore » and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.« less

Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by φC31 integrase.

PubMed

Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J

2011-11-01

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens

PubMed Central

Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

2015-01-01

Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies. PMID:26713728

The detailed 3D multi-loop aggregate/rosette chromatin architecture and functional dynamic organization of the human and mouse genomes.

PubMed

Knoch, Tobias A; Wachsmuth, Malte; Kepper, Nick; Lesnussa, Michael; Abuseiris, Anis; Ali Imam, A M; Kolovos, Petros; Zuin, Jessica; Kockx, Christel E M; Brouwer, Rutger W W; van de Werken, Harmen J G; van IJcken, Wilfred F J; Wendt, Kerstin S; Grosveld, Frank G

2016-01-01

The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture ( T2C ), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.

Influence of a family 29 carbohydrate binding module on the recombinant production of galactose oxidase in Pichia pastoris

PubMed Central

Mollerup, Filip; Master, Emma

2015-01-01

Herein, we report the extracellular expression of carbohydrate active fusion enzymes in Pichia pastoris. Particularly, CBM29-1-2 from Piromyces equi was separately fused to the N- and C-terminus of galactose 6-oxidase (GaO, D-galactose: oxygen 6-oxidoreductase, EC 1.1.13.9, CAZy family AA5) from Fusarium graminearum, generating CBM29-GaO and GaO-CBM29, respectively. P. pastoris was transformed with expression vectors encoding GaO, CBM29-GaO and GaO-CBM29, and the fusion proteins were expressed in both shake-flask and 2L bioreactor systems. Volumetric production yields and specific GaO activity increased when expression was performed in a bioreactor system compared to shake-flask cultivation. This was observed for both CBM29-GaO and GaO-CBM29, and is consistent with previous reports of GaO expression in P. pastoris (Spadiut et al., 2010; Anasontzis et al., 2014) [1], [2]. Fusion of CBM29 to the C-terminal of GaO (GaO-CBM29) resulted in a stable uniform protein at the expected calculated size (107 kDa) when analyzed with SDS-PAGE. By comparison, the expression of the N-terminal fusion protein (CBM29-GaO) was low, and two truncated versions of CBM29-GaO were coexpressed with the full-sized protein. Despite differences in protein yield, the specific GaO activity on galactose was not affected by CBM29 fusion to either the N- or C-terminus of the enzyme. A detailed description of the catalytic and physiochemical properties of CBM29-GaO and GaO-CBM29 is available in the parent publication (Mollerup et al., 2015) [3]. PMID:26858983

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

PubMed

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

2017-03-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production.

PubMed

Peters-Wendisch, P; Götker, S; Heider, S A E; Komati Reddy, G; Nguyen, A Q; Stansen, K C; Wendisch, V F

2014-12-20

The Gram-positive Corynebacterium glutamicum is auxotrophic for biotin. Besides the biotin uptake system BioYMN and the transcriptional regulator BioQ, this bacterium possesses functional enzymes for the last three reactions of biotin synthesis starting from pimeloyl-CoA. Heterologous expression of bioF from the Gram-negative Escherichia coli enabled biotin synthesis from pimelic acid added to the medium, but expression of bioF together with bioC and bioH from E. coli did not entail biotin prototrophy. Heterologous expression of bioWAFDBI from Bacillus subtilis encoding another biotin synthesis pathway in C. glutamicum allowed for growth in biotin-depleted media. Stable growth of the recombinant was observed without biotin addition for eight transfers to biotin-depleted medium while the empty vector control stopped growth after the first transfer. Expression of bioWAFDBI from B. subtilis in C. glutamicum strains overproducing the amino acids l-lysine and l-arginine, the diamine putrescine, and the carotenoid lycopene, respectively, enabled formation of these products under biotin-depleted conditions. Thus, biotin-prototrophic growth and production by recombinant C. glutamicum were achieved. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of a heat-stable and orally delivered recombinant M2e-expressing B. subtilis spore-based influenza vaccine.

PubMed

Zhao, Guangyu; Miao, Yu; Guo, Yan; Qiu, Hongjie; Sun, Shihui; Kou, Zhihua; Yu, Hong; Li, Junfeng; Chen, Yue; Jiang, Shibo; Du, Lanying; Zhou, Yusen

2014-01-01

Highly conserved ectodomain of influenza virus M2 protein (M2e) is an important target for the development of universal influenza vaccines. Today, the use of chemical or genetic fusion constructs have been undertaken to overcome the low immunogenicity of M2e in vaccine formulation. However, current M2e vaccines are neither orally delivered nor heat-stable. In this study, we evaluated the immune efficacy of an orally delivered recombinant M2e vaccine containing 3 molcules of M2e consensus sequence of influenza A viruses, termed RSM2e3. To accomplish this, CotB, a spore coat of Bacillus subtilis (B. subtilis), was used as a fusion partner, and heat-stable nonpathogenic B. subtilis spores were used as the carrier. Our results showed that CotB-M2e3 fusion had no effect on spore structure or function in the resultant recombinant RSM2e3 strain and that heterologous influenza virus M2e protein was successfully displayed on the surface of the recombinant RSM2e3 spore. Importantly, recombinant RSM2e3 spores elicited strong and long-term M2e-specific systemic and mucosal immune responses, completely protecting immunized mice from lethal challenge of A/PR/8/34(H1N1) influenza virus. Taken together, our study forms a solid basis for the development of a novel orally delivered and heat-stable influenza vaccine based on B. subtilis spore surface display.

A systems biology pipeline identifies new immune and disease related molecular signatures and networks in human cells during microgravity exposure

NASA Astrophysics Data System (ADS)

Mukhopadhyay, Sayak; Saha, Rohini; Palanisamy, Anbarasi; Ghosh, Madhurima; Biswas, Anupriya; Roy, Saheli; Pal, Arijit; Sarkar, Kathakali; Bagh, Sangram

2016-05-01

Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions.

Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

PubMed Central

Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

2015-01-01

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. PMID:26670247

A systems biology pipeline identifies new immune and disease related molecular signatures and networks in human cells during microgravity exposure.

PubMed

Mukhopadhyay, Sayak; Saha, Rohini; Palanisamy, Anbarasi; Ghosh, Madhurima; Biswas, Anupriya; Roy, Saheli; Pal, Arijit; Sarkar, Kathakali; Bagh, Sangram

2016-05-17

Microgravity is a prominent health hazard for astronauts, yet we understand little about its effect at the molecular systems level. In this study, we have integrated a set of systems-biology tools and databases and have analysed more than 8000 molecular pathways on published global gene expression datasets of human cells in microgravity. Hundreds of new pathways have been identified with statistical confidence for each dataset and despite the difference in cell types and experiments, around 100 of the new pathways are appeared common across the datasets. They are related to reduced inflammation, autoimmunity, diabetes and asthma. We have identified downregulation of NfκB pathway via Notch1 signalling as new pathway for reduced immunity in microgravity. Induction of few cancer types including liver cancer and leukaemia and increased drug response to cancer in microgravity are also found. Increase in olfactory signal transduction is also identified. Genes, based on their expression pattern, are clustered and mathematically stable clusters are identified. The network mapping of genes within a cluster indicates the plausible functional connections in microgravity. This pipeline gives a new systems level picture of human cells under microgravity, generates testable hypothesis and may help estimating risk and developing medicine for space missions.

Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents.

PubMed

Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

2015-12-08

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications.

Assessment of RNAi-induced silencing in banana (Musa spp.).

PubMed

Dang, Tuong Vi T; Windelinckx, Saskia; Henry, Isabelle M; De Coninck, Barbara; Cammue, Bruno P A; Swennen, Rony; Remy, Serge

2014-09-18

In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana. Using embryogenic cell suspension (ECS) transformed with ß-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using three intron-spliced hairpin RNA (ihpRNA) constructs containing gusAINT sequences of 299-nt, 26-nt and 19-nt, respectively. Their silencing potential was analysed in 2 different experimental set-ups. In the first, Agrobacterium-mediated co-transformation of banana ECS with a gusAINT containing vector and an ihpRNA construct resulted in a significantly reduced GUS enzyme activity 6-8 days after co-cultivation with either the 299-nt and 19-nt ihpRNA vectors. In the second approach, these ihpRNA constructs were transferred to stable GUS-expressing ECS and their silencing potential was evaluated in the regenerated in vitro plants. In comparison to control plants, transgenic plants transformed with the 299-nt gusAINT targeting sequence showed a 4.5 fold down-regulated gusA mRNA expression level, while GUS enzyme activity was reduced by 9 fold. Histochemical staining of plant tissues confirmed these findings. Northern blotting used to detect the expression of siRNA in the 299-nt ihpRNA vector transgenic in vitro plants revealed a negative relationship between siRNA expression and GUS enzyme activity. In contrast, no reduction in GUS activity or GUS mRNA expression occurred in the regenerated lines transformed with either of the two gusAINT oligo target sequences (26-nt and 19-nt). RNAi-induced silencing was achieved in banana, both at transient and stable level, resulting in significant reduction of gene expression and enzyme activity. The success of silencing was dependent on the targeted region of the target gene. The successful generation of transgenic ECS for second transformation with (an)other construct(s) can be of value for functional genomics research in banana.

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

USDA-ARS?s Scientific Manuscript database

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

Production of a yeast artificial chromosome for stable expression of a synthetic xylose isomerase-xylulokinase polyprotein in a fuel ethanol yeast strain

USDA-ARS?s Scientific Manuscript database

Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to utilize pentose sugars. A yeast artificial chromosome (YAC) was engineered to contain a polyprotein gene construct expressing xylos...

The ULT1 and ULT2 trxG genes play overlapping roles in Arabidopsis development and gene regulation

USDA-ARS?s Scientific Manuscript database

The epigenetic regulation of gene expression is critical for ensuring the proper deployment and stability of defined genome transcription programs at specific developmental stages. The cellular memory of stable gene expression states during animal and plant development is mediated by the opposing ac...

Validation of reference genes for gene expression studies in soybean aphid, Aphis glycines Matsumura

USDA-ARS?s Scientific Manuscript database

Quantitative real-time PCR (qRT-PCR) is a common tool for quantifying mRNA transcripts. To normalize results, a reference gene is mandatory. Aphis glycines is a significant soybean pest, yet gene expression and functional genomics studies are hindered by a lack of stable reference genes. We evalu...

Cardiac RNAi therapy using RAGE siRNA/deoxycholic acid-modified polyethylenimine complexes for myocardial infarction.

PubMed

Hong, Jueun; Ku, Sook Hee; Lee, Min Sang; Jeong, Ji Hoon; Mok, Hyejung; Choi, Donghoon; Kim, Sun Hwa

2014-08-01

Inflammatory response in myocardial ischemia-reperfusion injury plays a critical role in ventricular remodeling. To avoid deleterious effects of overwhelming inflammation, we blocked the expression of receptor for advanced glycation end-products (RAGE), a key mediator of the local and systemic inflammatory responses, via RNAi mechanism. Herein, a facial amphipathic deoxycholic acid-modified low molecular weight polyethylenimine (DA-PEI) was used as a siRNA delivery carrier to myocardium. The DA-PEI conjugate formed a stable complex with siRNA via electrostatic and hydrophobic interactions. The siRAGE/DA-PEI formulation having negligible toxicity could enhance intracellular delivery efficiency and successfully suppress RAGE expression both in vitro and in vivo. Furthermore, the cardiac administration of siRAGE/DA-PEI reduced apoptosis and inflammatory cytokine release, subsequently led to attenuation of left ventricular remodeling in rat myocardial infarction model. The potential therapeutic effects of RAGE gene silencing on myocardial ischemia-reperfusion injury may suggest that the siRAGE/DA-PEI delivery system can be considered as a promising strategy for treating myocardial infarction. Copyright © 2014 Elsevier Ltd. All rights reserved.

Role of Rayleigh numbers on characteristics of double diffusive salt fingers

NASA Astrophysics Data System (ADS)

Rehman, F.; Singh, O. P.

2018-05-01

Double diffusion convection, driven by two constituents of the fluid with different molecular diffusivity, is widely applied in oceanography and large number of other fields like astrophysics, geology, chemistry and metallurgy. In case of ocean, heat (T) and salinity (S) are the two components with varying diffusivity, where heat diffuses hundred times faster than salt. Component (T) stabilizes the system whereas components (S) destabilizes the system with overall density remains stable and forms the rising and sinking fingers known as salt fingers. Recent observations suggest that salt finger characteristics such as growth rates, wavenumber, and fluxes are strongly depending on the Rayleigh numbers as major driving force. In this paper, we corroborate this observation with the help of experiments, numerical simulations and linear theory. An eigenvalue expression for growth rate is derived from the linearized governing equations with explicit dependence on Rayleigh numbers, density stability ratio, Prandtl number and diffusivity ratio. Expressions for fastest growing fingers are also derived as a function various non-dimensional parameter. The predicted results corroborate well with the data reported from the field measurements, experiments and numerical simulations.

A Consideration of Stable Operating Power Limits of HVDC System Composed of Voltage Source Converters

NASA Astrophysics Data System (ADS)

Konishi, Hiroo; Takahashi, Choei; Kishibe, Hideto; Sato, Hiromichi

The stable operating power limits of a small scale HVDC system composed of voltage source converters (VSC-HVDC system) are analyzed with a simple model. The VSC-HVDC system could operate where the AC system must be somewhat larger in capacity than the VSC-HVDC system capacity. The stable operating power limits were between one and two times the SCR (short circuit ratio). When the inverter of the VSC-HVDC system was operated with lead reactive (capacitive) power control conditions, the stable operating limits were increased through AC voltage stabilization. When the inverter was a STATCOM operation, it could operate regardless of the SCR but regions within allowable AC voltage variations.

Temperature Dependent Characterization of Terahertz Vibrations of Explosives and Related Threat Materials

DTIC Science & Technology

2010-12-10

absorption spectra of 1,3,5,7- tetranitro-1,3,5,7-tetrazocane ( HMX ) polymorphs ,” Chem. Phys. Lett. 489(1-3), 48–53 (2010). 23. P. Main, R. E...The β polymorph is the most stable form of the four polymorphs (α,β,γ,δ) of HMX and the room temperature THz spectrum of β- HMX has been measured by...EXPRESS 27248 polymorph to be formed. The α- HMX and δ- HMX forms are described as stable above room temperature: α- HMX is stable from 377 K to 429 K [24

Stable isotope dimethyl labelling for quantitative proteomics and beyond

PubMed Central

Hsu, Jue-Liang; Chen, Shu-Hui

2016-01-01

Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644970

A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits.

PubMed

Estornell, Leandro Hueso; Orzáez, Diego; López-Peña, Lucas; Pineda, Benito; Antón, María Teresa; Moreno, Vicente; Granell, Antonio

2009-04-01

A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.

Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination

PubMed Central

Nochi, Tomonori; Takagi, Hidenori; Yuki, Yoshikazu; Yang, Lijun; Masumura, Takehiro; Mejima, Mio; Nakanishi, Ushio; Matsumura, Akiko; Uozumi, Akihiro; Hiroi, Takachika; Morita, Shigeto; Tanaka, Kunisuke; Takaiwa, Fumio; Kiyono, Hiroshi

2007-01-01

Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB) under the control of the endosperm-specific expression promoter 2.3-kb glutelin GluB-1 with codon usage optimization for expression in rice seed. An average of 30 μg of CTB per seed was stored in the protein bodies, which are storage organelles in rice. When mucosally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches and induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for >1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they require neither refrigeration (cold-chain management) nor a needle, these rice-based mucosal vaccines offer a highly practical and cost-effective strategy for orally vaccinating large populations against mucosal infections, including those that may result from an act of bioterrorism. PMID:17573530

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

PubMed Central

Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

2014-01-01

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

Hybrid Adeno-Associated Viral Vectors Utilizing Transposase-Mediated Somatic Integration for Stable Transgene Expression in Human Cells

PubMed Central

Zhang, Wenli; Solanki, Manish; Müther, Nadine; Ebel, Melanie; Wang, Jichang; Sun, Chuanbo; Izsvak, Zsuzsanna; Ehrhardt, Anja

2013-01-01

Recombinant adeno-associated viral (AAV) vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB) transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR) protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells. PMID:24116154

Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system.

PubMed

Lemaire, Géraldine; Mnif, Wissem; Pascussi, Jean-Marc; Pillon, Arnaud; Rabenoelina, Fanja; Fenet, Hélène; Gomez, Elena; Casellas, Claude; Nicolas, Jean-Claude; Cavaillès, Vincent; Duchesne, Marie-Josèphe; Balaguer, Patrick

2006-06-01

Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells.

PubMed

Xu, Kun; Zhang, Ting Ting; Wang, Ling; Zhang, Cun Fang; Zhang, Long; Ma, Li Xia; Xin, Ying; Ren, Chong Hua; Zhang, Zhi Qiang; Yan, Qiang; Martineau, Daniel; Zhang, Zhi Ying

2013-02-01

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8 kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8 kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin.

PubMed

Rovère, C; Barbero, P; Kitabgi, P

1996-05-10

The neuropeptide precursor proneurotensin/neuromedin N (pro-NT/NN) is mainly expressed and differentially processed in the brain and in the small intestine. We showed previously that rMTC 6-23 cells process pro-NT/NN with a pattern similar to brain tissue and increase pro-NT/NN expression in response to dexamethasone, and that PC12 cells also produce pro-NT/NN but are virtually unable to process it. In addition, PC12 cells were reported to be devoid of the prohormone convertases PC1 and PC2. The present study was designed to identify the proprotein convertase(s) (PC) involved in pro-NT/NN processing in rMTC 6-23 cells and to compare PC1- and PC2-transfected PC12 cells for their ability to process pro-NT/NN. rMTC 6-23 cells were devoid of PC1, PC4, and PC5 but expressed furin and PC2. Stable expression of antisense PC2 RNA in rMTC 6-23 cells led to a 90% decrease in PC2 protein levels that correlated with a > 80% reduction of pro-NT/NN processing. PC2 expression was stimulated by dexamethasone in a time- and concentration-dependent manner. Stable PC12/PC2 transfectants processed pro-NT/NN with a pattern similar to that observed in the brain and in rMTC 6-23 cells. In contrast, stable PC12/PC1 transfectants reproduced the pro-NT/NN processing pattern seen in the gut. We conclude that (i) PC2 is the major pro-NT/NN convertase in rMTC 6-23 cells; (ii) its expression is coregulated with that of pro-NT/NN in this cell line; and (iii) PC2 and PC1 differentially process pro-NT/NN with brain and intestinal phenotype, respectively.

Focus on ulcerative colitis: stable gastric pentadecapeptide BPC 157.

PubMed

Sikiric, P; Seiwerth, S; Rucman, R; Turkovic, B; Rokotov, D S; Brcic, L; Sever, M; Klicek, R; Radic, B; Drmic, D; Ilic, S; Kolenc, D; Stambolija, V; Zoricic, Z; Vrcic, H; Sebecic, B

2012-01-01

Stable gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, M.W. 1419) may be the new drug stable in human gastric juice, effective both in the upper and lower GI tract, and free of side effects. BPC 157, in addition to an antiulcer effect efficient in therapy of inflammatory bowel disease (IBD) (PL 14736) so far only tested in clinical phase II, has a very safe profile, and exhibited a particular wound healing effect. It also has shown to interact with the NO-system, providing endothelium protection and angiogenic effect, even in severely impaired conditions (i.e., it stimulated expression of early growth response 1 gene responsible for cytokine and growth factor generation and early extracellular matrix (collagen) formation (but also its repressor nerve growth factor 1-A binding protein-2)), important to counteract severe complications of advanced and poorly controlled IBD. Hopefully, the lessons from animal studies, particularly advanced intestinal anastomosis healing, reversed short bowel syndrome and fistula healing indicate BPC 157's high significance in further IBD therapy. Also, this supportive evidence (i.e., no toxic effect, limit test negative, LD1 not achieved, no side effect in trials) may counteract the problems commonly exercised in the use of peptidergic agents, particularly those used on a long-term basis.

Stable long-term blood formation by stem cells in murine steady-state hematopoiesis.

PubMed

Zavidij, Oksana; Ball, Claudia R; Herbst, Friederike; Oppel, Felix; Fessler, Sylvia; Schmidt, Manfred; von Kalle, Christof; Glimm, Hanno

2012-09-01

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis. Copyright © 2012 AlphaMed Press.

Decationized polyplexes as stable and safe carrier systems for improved biodistribution in systemic gene therapy

PubMed Central

Golombek, Susanne K.; Dakwar, George R.; Lou, Bo; Remaut, Katrien; Mastrobattista, Enrico; van Nostrum, Cornelus F.; Jahnen-Dechent, Wilhelm; Kiessling, Fabian; Braeckmans, Kevin; Lammers, Twan; Hennink, Wim E.

2014-01-01

Many polycation-based gene delivery vectors show high transfection in vitro, but their cationic nature generally leads to significant toxicity and poor in vivo performance which significantly hampers their clinical applicability. Unlike conventional polycation-based systems, decationized polyplexes are based on hydrophilic and neutral polymers. They are obtained by a 3-step process: charge-driven condensation followed by disulfide crosslinking stabilization and finally polyplex decationization. They consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). In the present paper the applicability of decationized polyplexes for systemic administration was evaluated. Cy5-labeled decationized polyplexes were evaluated for stability in plasma by fluorescence single particle tracking (fSPT), which technique showed stable size distribution for 48 h unlike its cationic counterpart. Upon the incubation of the polymers used for the formation of polyplexes with HUVEC cells, MTT assay showed excellent cytocompatibility of the neutral polymers. The safety was further demonstrated by a remarkable low teratogenicity and mortality activity of the polymers in a zebrafish assay, in great contrast with their cationic counterpart. Near infrared (NIR) dye-labeled polyplexes were evaluated for biodistribution and tumor accumulation by noninvasive optical imaging when administered systemically in tumor bearing mice. Decationized polyplexes exhibited an increased circulation time and higher tumor accumulation, when compared to their cationic precursors. Histology of tumors sections showed that decationized polyplexes induced reporter transgene expression in vivo. In conclusion, decationized polyplexes are a platform for safer polymeric vectors with improved biodistribution properties when systemically administered. PMID:25204289

Alpha-lipoic acid blocks HIV-1 LTR-dependent expression of hygromycin resistance in THP-1 stable transformants.

PubMed

Merin, J P; Matsuyama, M; Kira, T; Baba, M; Okamoto, T

1996-09-23

Gene expression of human immunodeficiency virus (HIV) depends on a host cellular transcription factors including nuclear factor-kappaB (NF-kappaB). The involvement of reactive oxygen intermediates (ROI) has been implicated as intracellular messengers in the inducible activation of NF-kappaB. In this study, we compared the efficacy of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), which are widely recognized NF-kappaB inhibitors. Here, we demonstrate that LA has a more potent activity in inhibiting NF-KappaB-mediated gene expression in THP-1 cells that have been stably transfected with a plasmid bearing a hygromycin B resistance gene under the control of HIV-1 long terminal repeat (LTR) promoter. The spontaneous activation of NF-kappaB in this cell culture system leads to expression of the hygromycin phosphotransferase gene hence rendering the cells resistance to hygromycin B. In this study, the effect of the test compounds against transcriptional activity of HIV-1 LTR was evaluated based on the degree of cellular toxicity due to the inhibitory activity on the expression of hygromycin B resistance gene in the presence of hygromycin B. We also found that 0.2 mM LA could cause 40% reduction in the HIV-1 expression from the TNF-alpha-stimulated OM 10.1, a cell line latently infected with HIV-1. On the other hand, 10 mM NAC was required to elicit the same effect. Furthermore, the initiation of HIV-1 induction by TNF-alpha was completely abolished by 1 mM LA. These findings confirm the involvement of ROI in NF-kappaB-mediated HIV gene expression as well as the efficacy of LA as a therapeutic regimen for HIV infection and acquired immunodeficiency syndrome (AIDS). Moreover, this study validates the applicability of our present assay system which we primarily designed for the screening of candidate drugs against HIV-1 gene expression.

Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

PubMed Central

Lan, Feng; Liu, Junwei; Narsinh, Kazim H.; Hu, Shijun; Han, Leng; Lee, Andrew S.; Karow, Marisa; Nguyen, Patricia K.; Nag, Divya; Calos, Michele P.; Robbins, Robert C.; Wu, Joseph C.

2012-01-01

Background Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. Conclusions The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types. PMID:22965984

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

PubMed

Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

2012-09-11

Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

The acute and temporary modulation of PERIOD genes by hydrocortisone in healthy subjects.

PubMed

Yurtsever, Türkan; Schilling, Thomas M; Kölsch, Monika; Turner, Jonathan D; Meyer, Jobst; Schächinger, Hartmut; Schote, Andrea B

2016-01-01

The physiological stress system and the circadian clock system communicate with each other at different signaling levels. The steroid hormone cortisol, the end-effector of the hypothalamus-pituitary-adrenal axis, is released in response to stress and acts as a mediator in circadian rhythms. We determined the effect of escalating cortisol doses on the expression of PERIOD genes (PER1, PER2 and PER3) in healthy subjects and analyzed whether the glucocorticoid receptor (GR) is involved in the cortisol-mediated PERIOD gene expression. Forty participants (50% males and 50% females) were randomly assigned to groups receiving a saline placebo solution or 3 mg, 6 mg, 12 mg and 24 mg of hydrocortisone. Blood was drawn every 15 min to measure quantitative gene expression of PER1, PER2 and PER3. A potential role of the GR was determined by an ex vivo study stimulating whole blood with hydrocortisone and RU486 (a GR antagonist). As a result, moderate doses of hydrocortisone produced an acute and temporary induction of PER1 and PER3 mRNA levels, whereas PER2 was not responsive to the hormone administration. The cortisol-dependent induction of PER1 was blocked by the GR antagonist in whole blood after treatment with hydrocortisone and RU486 ex vivo. In conclusion, acute pharmacological stress modulated the expression of PER1 and PER3 in whole blood temporarily in our short-term sampling design, suggesting that these circadian genes mediate stable molecular mechanisms in the periphery.

Nontransgenic Genome Modification in Plant Cells1[W][OA

PubMed Central

Marton, Ira; Zuker, Amir; Shklarman, Elena; Zeevi, Vardit; Tovkach, Andrey; Roffe, Suzy; Ovadis, Marianna; Tzfira, Tzvi; Vainstein, Alexander

2010-01-01

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods. PMID:20876340

Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

PubMed Central

Singh, Harjeet; Figliola, Matthew J.; Dawson, Margaret J.; Olivares, Simon; Zhang, Ling; Yang, Ge; Maiti, Sourindra; Manuri, Pallavi; Senyukov, Vladimir; Jena, Bipulendu; Kebriaei, Partow; Champlin, Richard E.; Huls, Helen; Cooper, Laurence J. N.

2013-01-01

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion. PMID:23741305

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

PubMed

de Vries, R P; van de Vondervoort, P J I; Hendriks, L; van de Belt, M; Visser, J

2002-09-01

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

RGS10 exerts a neuroprotective role through the PKA/c-AMP response-element (CREB) pathway in dopaminergic neuron-like cells

PubMed Central

Lee, Jae-Kyung; Chung, Jaegwon; Druey, Kirk M.; Tansey, Malú G.

2012-01-01

Regulator of G-protein signaling-10 (RGS10) is a GTPase activating protein (GAP) for Gαi/q/z subunits that is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain. Previously, we reported that RGS10-null mice display increased vulnerability to chronic systemic inflammation-induced degeneration of nigral dopaminergic (DA) neurons. Given that RGS10 is expressed in DA neurons, we investigated the extent to which RGS10 regulates cell survival under conditions of inflammatory stress. Because of the inherent limitations associated with use of primary DA neurons for biochemical analyses, we employed a well-characterized ventral mesencephalon DA neuroblastoma cell line (MN9D) for our studies. We found that stable over-expression of RGS10 rendered them resistant to TNF-induced cytotoxicity; whereas MN9D cells expressing mutant RGS10-S168A (which is resistant to phosphorylation by protein kinase A (PKA) at a serine residue that promotes its nuclear translocation) showed similar sensitivity to TNF as the parental MN9D cells. Using biochemical and pharmacological approaches, we identified protein kinase A (PKA) and the downstream phospho-cAMP response element-binding (CREB) signaling pathway (and ruled out ERK 1/2, JNK, and NFkB) as key mediators of the neuroprotective effect of RGS10 against inflammatory stress. PMID:22564151

Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

PubMed

Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

2000-08-01

The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides1[W][OPEN

PubMed Central

Muthukumar, Balasubramaniam; Joyce, Blake L.; Elless, Mark P.; Stewart, C. Neal

2013-01-01

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid ‘C-fern Express’ (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using β-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns. PMID:23933990

RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers

PubMed Central

Tan, Jean-Marie; Payne, Elizabeth J.; Lin, Lynlee L.; Sinnya, Sudipta; Raphael, Anthony P.; Lambie, Duncan; Frazer, Ian H.; Dinger, Marcel E.; Soyer, H. Peter

2017-01-01

Identification of appropriate reference genes (RGs) is critical to accurate data interpretation in quantitative real-time PCR (qPCR) experiments. In this study, we have utilised next generation RNA sequencing (RNA-seq) to analyse the transcriptome of a panel of non-melanoma skin cancer lesions, identifying genes that are consistently expressed across all samples. Genes encoding ribosomal proteins were amongst the most stable in this dataset. Validation of this RNA-seq data was examined using qPCR to confirm the suitability of a set of highly stable genes for use as qPCR RGs. These genes will provide a valuable resource for the normalisation of qPCR data for the analysis of non-melanoma skin cancer. PMID:28852586

[Circular RNA in human disease and their potential clinic significance].

PubMed

Chen, Yonghua; Li, Cheng; Tan, Chunlu; Mai, Gang; Liu, Xubao

2017-02-10

Circular RNAs (circ RNAs) are a novel type of RNA that, unlike linear RNAs, form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome. They share a stable structure, high expression and often exhibit tissue/developmental-stage-specific expression. Emerging evidence indicates that circRNAs might play important roles in human disease, such as cancer, neurological disorders and atherosclerotic vascular disease risk. The huge potentials of circRNAs are recently being discovered from the laboratory to the clinic. CircRNAs might be developed as a potential novel and stable biomarker and potential drugs used in disease diagnosis and treatment. Here, we review the current understanding of the roles of circRNAs in human disease and their potential clinic significance in disease.

RNAi-dependent and -independent antiviral phenotypes of chromosomally integrated shRNA clones: role of VASP in respiratory syncytial virus growth.

PubMed

Musiyenko, Alla; Bitko, Vira; Barik, Sailen

2007-07-01

Stable RNA interference (RNAi) is commonly achieved by recombinant expression of short hairpin RNA (shRNA). To generate virus-resistant cell lines, we cloned a shRNA cassette against the phosphoprotein gene of respiratory syncytial virus (RSV) into a polIII-driven plasmid vector. Analysis of individual stable transfectants showed a spectrum of RSV resistance correlating with the levels of shRNA expressed from different chromosomal locations. Interestingly, resistance in a minority of clones was due to mono-allelic disruption of the cellular gene for vasodilator-stimulated phosphoprotein (VASP). Thus, pure clones of chromosomally integrated DNA-directed RNAi can exhibit gene disruption phenotypes resembling but unrelated to RNAi.

Prolonged striatal disinhibition as a chronic animal model of tic disorders.

PubMed

Vinner, Esther; Israelashvili, Michal; Bar-Gad, Izhar

2017-12-01

Experimental findings and theoretical models have associated Tourette syndrome with abnormal striatal inhibition. The expression of tics, the hallmark symptom of this disorder, has been transiently induced in non-human primates and rodents by the injection of GABA A antagonists into the striatum, leading to temporary disinhibition. The novel chronic model of tic expression utilizes mini-osmotic pumps implanted subcutaneously in the rat's back for prolonged infusion of bicuculline into the dorsolateral striatum. Tics were expressed on the contralateral side to the infusion over a period of multiple days. Tic expression was stable, and maintained similar properties throughout the infusion period. Electrophysiological recordings revealed the existence of tic-related local field potential spikes and individual neuron activity changes that remained stable throughout the infusion period. The striatal disinhibition model provides a unique combination of face validity (tic expression) and construct validity (abnormal striatal inhibition) but is limited to sub-hour periods. The new chronic model extends the period of tic expression to multiple days and thus enables the study of tic dynamics and the effects of behavior and pharmacological agents on tic expression. The chronic model provides similar behavioral and neuronal correlates of tics as the acute striatal disinhibition model but over prolonged periods of time, thus providing a unique, basal ganglia initiated model of tic expression. Chronic expression of symptoms is the key to studying the time varying properties of Tourette syndrome and the effects of multiple internal and external factors on this disorder. Copyright © 2017 Elsevier B.V. All rights reserved.

Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells.

PubMed

Wang, Linli; Chen, Yuehua; Guan, Chunyan; Zhao, Zhiju; Li, Qiang; Yang, Jianguo; Mo, Jian; Wang, Bin; Wu, Wei; Yang, Xiaohui; Song, Libing; Li, Jun

2017-11-02

Because the lack of an induced pluripotent stem cell (iPSC) induction system with optimal safety and efficiency limits the application of these cells, development of such a system is important. To create such an induction system, we screened a variety of reprogrammed plasmid combinations and multiple compounds and then verified the system's feasibility using urine cells from different individuals. We also compared large-scale iPSC chromosomal variations and expression of genes associated with genomic stability between this system and the traditional episomal system using karyotype and quantitative reverse transcription polymerase chain reaction analyses. We developed a high-efficiency episomal system, the 6F/BM1-4C system, lacking tumorigenic factors for human urine-derived cell (hUC) reprogramming. This system includes six low-risk factors (6F), Oct4, Glis1, Klf4, Sox2, L-Myc, and the miR-302 cluster. Transfected hUCs were treated with four compounds (4C), inhibitor of lysine-demethylase1, methyl ethyl ketone, glycogen synthase kinase 3 beta, and histone deacetylase, within a short time period. Comparative analysis revealed significantly decreased chromosomal variation in iPSCs and significantly increased Sirt1 expression compared with iPSCs induced using the traditional episomal system. The 6F/BM1-4C system effectively induces reprogramming of urine cells in samples obtained from different individuals. iPSCs induced using the 6F/BM1-4C system are more stable at the cytogenetic level and have potential value for clinical application.

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. This result provides a new insight into the investigation of the mechanism of robust patterning in biological systems composed of individual elements. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

2006-11-01

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

Gene transfer and expression in plants.

PubMed

Lorence, Argelia; Verpoorte, Robert

2004-01-01

Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them. This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics). Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).

Altered Innate Defenses in the Neonatal Gastrointestinal Tract in Response to Colonization by Neuropathogenic Escherichia coli

PubMed Central

Birchenough, George M. H.; Johansson, Malin E. V.; Stabler, Richard A.; Dalgakiran, Fatma; Hansson, Gunnar C.; Wren, Brendan W.; Luzio, J. Paul

2013-01-01

Two-day-old (P2), but not 9-day-old (P9), rat pups are susceptible to systemic infection following gastrointestinal colonization by Escherichia coli K1. Age dependency reflects the capacity of colonizing K1 to translocate from gastrointestinal (GI) tract to blood. A complex GI microbiota developed by P2, showed little variation over P2 to P9, and did not prevent stable K1 colonization. Substantial developmental expression was observed over P2 to P9, including upregulation of genes encoding components of the small intestinal (α-defensins Defa24 and Defa-rs1) and colonic (trefoil factor Tff2) mucus barrier. K1 colonization modulated expression of these peptides: developmental expression of Tff2 was dysregulated in P2 tissues and was accompanied by a decrease in mucin Muc2. Conversely, α-defensin genes were upregulated in P9 tissues. We propose that incomplete development of the mucus barrier during early neonatal life and the capacity of colonizing K1 to interfere with mucus barrier maturation provide opportunities for neuropathogen translocation into the bloodstream. PMID:23798529

Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system.

PubMed

Sugano, Shigeo S; Suzuki, Hiroko; Shimokita, Eisuke; Chiba, Hirofumi; Noji, Sumihare; Osakabe, Yuriko; Osakabe, Keishi

2017-04-28

Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

Biodegradable DNA Nanoparticles that Provide Widespread Gene Delivery in the Brain

PubMed Central

Mastorakos, Panagiotis; Song, Eric; Zhang, Clark; Berry, Sneha; Park, Hee Won; Kim, Young Eun; Park, Jong Sung; Lee, Seulki; Suk, Jung Soo; Hanes, Justin

2016-01-01

Successful gene therapy of neurological disorders is predicated on achieving widespread and uniform transgene expression throughout the affected disease area in the brain. However, conventional gene vectors preferentially travel through low-resistance perivascular spaces and/or are confined to the administration site even with the aid of a pressure-driven flow provided by convection-enhanced delivery. Biodegradable DNA nanoparticles offer a safe gene delivery platform devoid of adverse effects associated with virus-based or synthetic non-biodegradable systems. Using a state-of-the-art biodegradable polymer, poly(β-amino ester), we engineered colloidally stable sub-100 nm DNA nanoparticles coated with a non-adhesive polyethylene glycol corona that are able to avoid the adhesive and steric hindrances imposed by the extracellular matrix. Following convection enhanced delivery, these brain-penetrating nanoparticles were able to homogeneously distribute throughout the rodent striatum and mediate widespread and high-level transgene expression. These nanoparticles provide a biodegradable DNA nanoparticle platform enabling uniform transgene expression patterns in vivo and hold promise for the treatment of neurological diseases. PMID:26680637

Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies

PubMed Central

Fukuyama, Satoshi; Katsura, Hiroaki; Zhao, Dongming; Ozawa, Makoto; Ando, Tomomi; Shoemaker, Jason E.; Ishikawa, Izumi; Yamada, Shinya; Neumann, Gabriele; Watanabe, Shinji; Kitano, Hiroaki; Kawaoka, Yoshihiro

2015-01-01

Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours (‘Color-flu’ viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis. PMID:25807527

High level expression and immunochemical characterization of botulinum neurotoxin type F light chain.

PubMed

Chauhan, Ritika; Chauhan, Vinita; Rao, Mula Kameshwar; Chaudhary, Dilip; Bhagyawant, Sameer; Dhaked, Ram Kumar

2018-06-01

Botulinum neurotoxins (BoNTs) are the most toxic biological substances known. Their potential use as biological warfare agent results in their classification as category A biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. Presently, there are no approved detection system and pharmacological treatments for BoNT intoxication. Although a toxoid vaccine is available for immuno-prophylaxis, vaccines cannot reverse the effect of pre-translocated toxin. Direct handling of the live BoNTs for developing detection and therapeutics may pose fatal danger. This concern was addressed by purifying the recombinant catalytically active light chain of BoNT/F. BoNT/F-LC gene was amplified from the genomic DNA using specifically designed primers and expressed in Escherichia coli. Expression and purification profile were optimized under different conditions for biologically active light chain production. Specific polyclonal antibodies generated against type F illustrates in vivo neutralization in mice and rabbit. These antibodies play key role in conceiving the development of high throughput SPR based detection system which is a highly precise label free technique for protein interaction analysis. The presented work is first of its kind, signifying the production of highly stable and active rBoNT/F-LC and its immunochemical characterization. The study aids in paving the path towards developing a persistent detection system as well as in presenting comprehended scheme for in vitro small molecule therapeutics analysis. Copyright © 2018 Elsevier Inc. All rights reserved.

In1-ghrelin, a splice variant of ghrelin gene, is associated with the evolution and aggressiveness of human neuroendocrine tumors: Evidence from clinical, cellular and molecular parameters.

PubMed

Luque, Raul M; Sampedro-Nuñez, Miguel; Gahete, Manuel D; Ramos-Levi, Ana; Ibáñez-Costa, Alejandro; Rivero-Cortés, Esther; Serrano-Somavilla, Ana; Adrados, Magdalena; Culler, Michael D; Castaño, Justo P; Marazuela, Mónica

2015-08-14

Ghrelin system comprises a complex family of peptides, receptors (GHSRs), and modifying enzymes [e.g. ghrelin-O-acyl-transferase (GOAT)] that control multiple pathophysiological processes. Aberrant alternative splicing is an emerging cancer hallmark that generates altered proteins with tumorigenic capacity. Indeed, In1-ghrelin and truncated-GHSR1b splicing variants can promote development/progression of certain endocrine-related cancers. Here, we determined the expression levels of key ghrelin system components in neuroendocrine tumor (NETs) and explored their potential functional role. Twenty-six patients with NETs were prospectively/retrospectively studied [72 samples from primary and metastatic tissues (30 normal/42 tumors)] and clinical data were obtained. The role of In1-ghrelin in aggressiveness was studied in vitro using NET cell lines (BON-1/QGP-1). In1-ghrelin, GOAT and GHSR1a/1b expression levels were elevated in tumoral compared to normal/adjacent tissues. Moreover, In1-ghrelin, GOAT, and GHSR1b expression levels were positively correlated within tumoral, but not within normal/adjacent samples, and were higher in patients with progressive vs. with stable/cured disease. Finally, In1-ghrelin increased aggressiveness (e.g. proliferation/migration) of NET cells. Altogether, our data strongly suggests a potential implication of ghrelin system in the pathogenesis and/or clinical outcome of NETs, and warrant further studies on their possible value for the future development of molecular biomarkers with diagnostic/prognostic/therapeutic value.

In1-ghrelin, a splice variant of ghrelin gene, is associated with the evolution and aggressiveness of human neuroendocrine tumors: Evidence from clinical, cellular and molecular parameters

PubMed Central

Gahete, Manuel D.; Ramos-Levi, Ana; Ibáñez-Costa, Alejandro; Rivero-Cortés, Esther; Serrano-Somavilla, Ana; Adrados, Magdalena; Culler, Michael D.; Castaño, Justo P.; Marazuela, Mónica

2015-01-01

Ghrelin system comprises a complex family of peptides, receptors (GHSRs), and modifying enzymes [e.g. ghrelin-O-acyl-transferase (GOAT)] that control multiple pathophysiological processes. Aberrant alternative splicing is an emerging cancer hallmark that generates altered proteins with tumorigenic capacity. Indeed, In1-ghrelin and truncated-GHSR1b splicing variants can promote development/progression of certain endocrine-related cancers. Here, we determined the expression levels of key ghrelin system components in neuroendocrine tumor (NETs) and explored their potential functional role. Twenty-six patients with NETs were prospectively/retrospectively studied [72 samples from primary and metastatic tissues (30 normal/42 tumors)] and clinical data were obtained. The role of In1-ghrelin in aggressiveness was studied in vitro using NET cell lines (BON-1/QGP-1). In1-ghrelin, GOAT and GHSR1a/1b expression levels were elevated in tumoral compared to normal/adjacent tissues. Moreover, In1-ghrelin, GOAT, and GHSR1b expression levels were positively correlated within tumoral, but not within normal/adjacent samples, and were higher in patients with progressive vs. with stable/cured disease. Finally, In1-ghrelin increased aggressiveness (e.g. proliferation/migration) of NET cells. Altogether, our data strongly suggests a potential implication of ghrelin system in the pathogenesis and/or clinical outcome of NETs, and warrant further studies on their possible value for the future development of molecular biomarkers with diagnostic/prognostic/therapeutic value. PMID:26124083

Seed specific expression and analysis of recombinant human adenosine deaminase (hADA) in three host plant species.

PubMed

Doshi, Ketan M; Loukanina, Natalia N; Polowick, Patricia L; Holbrook, Larry A

2016-10-01

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26 U/g) followed by pea (3.23 U/g) and Nicotiana benthamiana (1.69 U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

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

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of themore » TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.« less

Shortening tobacco life cycle accelerates functional gene identification in genomic research.

PubMed

Ning, G; Xiao, X; Lv, H; Li, X; Zuo, Y; Bao, M

2012-11-01

Definitive allocation of function requires the introduction of genetic mutations and analysis of their phenotypic consequences. Novel, rapid and convenient techniques or materials are very important and useful to accelerate gene identification in functional genomics research. Here, over-expression of PmFT (Prunus mume), a novel FT orthologue, and PtFT (Populus tremula) lead to shortening of the tobacco life cycle. A series of novel short life cycle stable tobacco lines (30-50 days) were developed through repeated self-crossing selection breeding. Based on the second transformation via a gusA reporter gene, the promoter from BpFULL1 in silver birch (Betula pendula) and the gene (CPC) from Arabidopsis thaliana were effectively tested using short life cycle tobacco lines. Comparative analysis among wild type, short life cycle tobacco and Arabidopsis transformation system verified that it is optional to accelerate functional gene studies by shortening host plant material life cycle, at least in these short life cycle tobacco lines. The results verified that the novel short life cycle transgenic tobacco lines not only combine the advantages of economic nursery requirements and a simple transformation system, but also provide a robust, effective and stable host system to accelerate gene analysis. Thus, shortening tobacco life cycle strategy is feasible to accelerate heterologous or homologous functional gene identification in genomic research. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

The LIM homeobox protein mLIM3/Lhx3 induces expression of the prolactin gene by a Pit-1/GHF-1-independent pathway in corticotroph AtT20 cells.

PubMed

Girardin, S E; Benjannet, S; Barale, J C; Chrétien, M; Seidah, N G

1998-07-24

mLIM3, a member of the LIM homeobox family, was recently demonstrated to be critical for proliferation and differentiation of the pituitary cell lineage. Using a pool of degenerate oligonucleotides we determined the DNA sequence ANNAGGAAA(T/C)GA(CIG)AA as the set preferentially recognized by mLIM3. A nearly identical sequence is found in the prolactin (PRL) promoter, within a 15-mer stretch from nucleotides (nts) -218 to -204 which is highly conserved between human, rat, and bovine. In order to test the hypothesis of a transcriptional effect of mLIM3 on the prolactin promoter, stable transfectants of mLIM3 cDNA in AtT20 tumor cells revealed that PRL mRNA expression was induced in 3 separate stable clones. Gel retardation experiments performed using nuclear extracts isolated from one of the AtT20/mLIM3 stable transfectants revealed affinity towards the 15-mer element of the PRL promoter. From these results, we propose that the PRL promoter element (nts -218 to -204) could be functional in vivo. Finally, we demonstrate that in AtT20 cells prolactin mRNA expression is not induced by the Pit-1/GHF-1 pathway and that growth hormone mRNA is not detected concomitantly with prolactin. We conclude that mLIM3 may play a key role in inducing PRL gene expression in lactotrophs by binding to a conserved motif close to a Pit-1/GHF-1 site within the proximal PRL promoter.

Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome

PubMed Central

Danger, Richard; Royer, Pierre-Joseph; Reboulleau, Damien; Durand, Eugénie; Loy, Jennifer; Tissot, Adrien; Lacoste, Philippe; Roux, Antoine; Reynaud-Gaubert, Martine; Gomez, Carine; Kessler, Romain; Mussot, Sacha; Dromer, Claire; Brugière, Olivier; Mornex, Jean-François; Guillemain, Romain; Dahan, Marcel; Knoop, Christiane; Botturi, Karine; Foureau, Aurore; Pison, Christophe; Koutsokera, Angela; Nicod, Laurent P.; Brouard, Sophie; Magnan, Antoine; Jougon, J.

2018-01-01

Bronchiolitis obliterans syndrome (BOS), the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group), and 26 samples at or after BOS diagnosis (diagnosis group). An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group). We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A), and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01) and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS. PMID:29375549

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

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

Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction.more » The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids. -- Highlights: Black-Right-Pointing-Pointer Cultured preadipocytes were transfected with sense lipocalin-type PGD synthase. Black-Right-Pointing-Pointer The cloned transfectants had a higher ability to synthesize PGD{sub 2} and PGJ{sub 2} series. Black-Right-Pointing-Pointer The sustained expression of sense L-PGDS hampered the storage of fats. Black-Right-Pointing-Pointer Adipogenesis program was suppressed independently of endogenous prostanoids.« less

Towards a General Theory of Extremes for Observables of Chaotic Dynamical Systems.

PubMed

Lucarini, Valerio; Faranda, Davide; Wouters, Jeroen; Kuna, Tobias

2014-01-01

In this paper we provide a connection between the geometrical properties of the attractor of a chaotic dynamical system and the distribution of extreme values. We show that the extremes of so-called physical observables are distributed according to the classical generalised Pareto distribution and derive explicit expressions for the scaling and the shape parameter. In particular, we derive that the shape parameter does not depend on the chosen observables, but only on the partial dimensions of the invariant measure on the stable, unstable, and neutral manifolds. The shape parameter is negative and is close to zero when high-dimensional systems are considered. This result agrees with what was derived recently using the generalized extreme value approach. Combining the results obtained using such physical observables and the properties of the extremes of distance observables, it is possible to derive estimates of the partial dimensions of the attractor along the stable and the unstable directions of the flow. Moreover, by writing the shape parameter in terms of moments of the extremes of the considered observable and by using linear response theory, we relate the sensitivity to perturbations of the shape parameter to the sensitivity of the moments, of the partial dimensions, and of the Kaplan-Yorke dimension of the attractor. Preliminary numerical investigations provide encouraging results on the applicability of the theory presented here. The results presented here do not apply for all combinations of Axiom A systems and observables, but the breakdown seems to be related to very special geometrical configurations.

Towards a General Theory of Extremes for Observables of Chaotic Dynamical Systems

NASA Astrophysics Data System (ADS)

Lucarini, Valerio; Faranda, Davide; Wouters, Jeroen; Kuna, Tobias

2014-02-01

In this paper we provide a connection between the geometrical properties of the attractor of a chaotic dynamical system and the distribution of extreme values. We show that the extremes of so-called physical observables are distributed according to the classical generalised Pareto distribution and derive explicit expressions for the scaling and the shape parameter. In particular, we derive that the shape parameter does not depend on the chosen observables, but only on the partial dimensions of the invariant measure on the stable, unstable, and neutral manifolds. The shape parameter is negative and is close to zero when high-dimensional systems are considered. This result agrees with what was derived recently using the generalized extreme value approach. Combining the results obtained using such physical observables and the properties of the extremes of distance observables, it is possible to derive estimates of the partial dimensions of the attractor along the stable and the unstable directions of the flow. Moreover, by writing the shape parameter in terms of moments of the extremes of the considered observable and by using linear response theory, we relate the sensitivity to perturbations of the shape parameter to the sensitivity of the moments, of the partial dimensions, and of the Kaplan-Yorke dimension of the attractor. Preliminary numerical investigations provide encouraging results on the applicability of the theory presented here. The results presented here do not apply for all combinations of Axiom A systems and observables, but the breakdown seems to be related to very special geometrical configurations.

Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator

USDA-ARS?s Scientific Manuscript database

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. In this study, expres...

In silico selection of expression reference genes with demonstrated stability in barley among a diverse set of tissues and cultivars

USDA-ARS?s Scientific Manuscript database

Premise of the study: Reference genes are selected based on the assumption of temporal and spatial expression stability and on their widespread use in model species. They are often used in new target species without validation, presumed as stable. For barley, reference gene validation is lacking, bu...

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation.

PubMed

Fustiñana, Maria Sol; Ariel, Pablo; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

2010-09-01

Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

Hyaluronic Acid-Based pH-Sensitive Polymer-Modified Liposomes for Cell-Specific Intracellular Drug Delivery Systems.

PubMed

Miyazaki, Maiko; Yuba, Eiji; Hayashi, Hiroshi; Harada, Atsushi; Kono, Kenji

2018-01-17

For the enhancement of therapeutic effects and reduction of side effects derived from anticancer drugs in cancer chemotherapy, it is imperative to develop drug delivery systems with cancer-specificity and controlled release function inside cancer cells. pH-sensitive liposomes are useful as an intracellular drug delivery system because of their abilities to transfer their contents into the cell interior through fusion or destabilization of endosome, which has weakly acidic environment. We earlier reported liposomes modified with various types of pH-sensitive polymers based on synthetic polymers and biopolymers as vehicles for intracellular drug delivery systems. In this study, hyaluronic acid (HA)-based pH-sensitive polymers were designed as multifunctional polymers having not only pH-sensitivity but also targeting properties to cells expressing CD44, which is known as a cancer cell surface marker. Carboxyl group-introduced HA derivatives of two types, MGlu-HA and CHex-HA, which have a more hydrophobic side chain structure than that of MGlu-HA, were synthesized by reaction with various dicarboxylic anhydrides. These polymer-modified liposomes were stable at neutral pH, but showed content release under weakly acidic conditions. CHex-HA-modified liposomes delivered their contents into CD44-expressing cells more efficiently than HA-modified or MGlu-HA-modified liposomes or unmodified liposomes, whereas the same liposomes were taken up only slightly by cells expressing CD44 proteins less. Competition assay using free HA or other polymers revealed that HA derivative-modified liposomes might be recognized by CD44. Therefore, HA-derivative-modified liposomes are useful as cell-specific intracellular drug delivery systems.

Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.

PubMed

Mort, Richard Lester; Ford, Matthew Jonathan; Sakaue-Sawano, Asako; Lindstrom, Nils Olof; Casadio, Angela; Douglas, Adam Thomas; Keighren, Margaret Anne; Hohenstein, Peter; Miyawaki, Atsushi; Jackson, Ian James

2014-01-01

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes-there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.

Free radical scavenging activities of yellow gentian (Gentiana lutea L.) measured by electron spin resonance.

PubMed

Kusar, A; Zupancic, A; Sentjurc, M; Baricevic, D

2006-10-01

Yellow gentian (Gentiana lutea L.) is a herbal species with a long-term use in traditional medicine due to its digestive and stomachic properties. This paper presents an investigation of the free radical scavenging activity of methanolic extracts of yellow gentian leaves and roots in two different systems using electron spin resonance (ESR) spectrometry. Assays were based on the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the superoxide radicals (O2*-) generated by the xanthine/xanthine oxidase (X/XO) system. The results of gentian methanolic extracts were compared with the antioxidant capacity of synthetic antioxidant butylated hydroxyanisole (BHA). This study proves that yellow gentian leaves and roots exhibit considerable antioxidant properties, expressed either by their capability to scavenge DPPH or superoxide radicals.

Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.

PubMed

Hardy, Melissa E; Ross, Louis V; Chien, Chi-Bin

2007-11-01

Misexpression of genes in a temporally and spatially controlled fashion is an important tool for assessing gene function during development. Because few tissue-specific promoters have been identified in zebrafish, inducible systems such as the Cre/LoxP and Tet repressor systems are of limited utility. Here we describe a new method of misexpression: local heat shock using a modified soldering iron. Zebrafish carrying transgenes under the control of a heat shock promoter (hsp70) are focally heated with the soldering iron to induce gene expression in a small area of the embryo. We have validated this method in three stable transgenic lines and at three developmental timepoints. Local heat shock is a fast, easy, and inexpensive method for gene misexpression. Copyright 2007 Wiley-Liss, Inc.

[Establishment and characterization of a new carcinoma cell line from uterine cervix of Uyghur women].

PubMed

Zhang, Lu; Aerziguli, Tursun; Guzalnur, Abliz

2012-04-01

To establish a uterine cervical carcinoma cell line of Uyghur ethnical background and to evaluate the related biological characteristics for future biomedical investigations of diseases in the Uyghur population. Poorly-differentiated squamous cell carcinoma specimens of Uyghur patients were obtained and cultured in vitro by enzymatic digestion method, followed by continuous passaging to reach a stable growth determined by cell viability and growth curve. Morphological study, cell cycling and chromosomal analysis were performed. Tumorigenesis study was conducted by inoculation of nude mice. Biomarker (CK17, CD44, Ki-67, CK14 and vimentin) expression was detected by immunofluorescence and immunocytochemical techniques. A cervical carcinoma cell line was successfully established and maintained for 12 months through 70 passages. The cell line had a stable growth with a population doubling time of 51.9 h. Flask method and double agar-agar assay showed that the cell line had colony-forming rates of 32.5% and 15.6%, respectively. Ultrastructural evaluation demonstrated numerous cell surface protrusions or microvilli, a large number of rod-shape structures in cytoplasm, typical desmosomes and nuclear atypia. Chromosomal analysis revealed human karyotype with the number of chromosomes per cell varying from 32 - 97 with a majority of 54 - 86 (60.3%). Xenogeneic tumors formed in nude mice showed histological structures identical to those of the primary tumor. The cells had high expression of CK17, CD44, Ki-67 and vimentin but no CK14 expression. A cervical carcinoma cell line from a female Uyghur patient is successfully established. The cell line has the characteristics of human cervical squamous cell carcinoma, and it is stable with maintaining the characteristic biological and morphological features in vitro for more than 12 months, therefore, qualified as a stable cell line for further biomedical research.

Modeling coexistence of oscillation and Delta/Notch-mediated lateral inhibition in pancreas development and neurogenesis.

PubMed

Tiedemann, Hendrik B; Schneltzer, Elida; Beckers, Johannes; Przemeck, Gerhard K H; Hrabě de Angelis, Martin

2017-10-07

During pancreas development, Neurog3 positive endocrine progenitors are specified by Delta/Notch (D/N) mediated lateral inhibition in the growing ducts. During neurogenesis, genes that determine the transition from the proneural state to neuronal or glial lineages are oscillating before their expression is sustained. Although the basic gene regulatory network is very similar, cycling gene expression in pancreatic development was not investigated yet, and previous simulations of lateral inhibition in pancreas development excluded by design the possibility of oscillations. To explore this possibility, we developed a dynamic model of a growing duct that results in an oscillatory phase before the determination of endocrine progenitors by lateral inhibition. The basic network (D/N + Hes1 + Neurog3) shows scattered, stable Neurog3 expression after displaying transient expression. Furthermore, we included the Hes1 negative feedback as previously discussed in neurogenesis and show the consequences for Neurog3 expression in pancreatic duct development. Interestingly, a weakened HES1 action on the Hes1 promoter allows the coexistence of stable patterning and oscillations. In conclusion, cycling gene expression and lateral inhibition are not mutually exclusive. In this way, we argue for a unified mode of D/N mediated lateral inhibition in neurogenic and pancreatic progenitor specification. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.