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Sample records for cell-free expression system

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

PubMed

Caschera, Filippo

2017-06-01

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

PubMed

Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

2015-06-01

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. Copyright © 2015 Elsevier Inc. All rights reserved.
Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

PubMed Central

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

2012-01-01

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

PubMed

He, Mingyue

2011-04-30

Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.
Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

PubMed

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

2013-02-10

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

PubMed

He, Mingyue

2008-01-01

Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.
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
Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

PubMed Central

Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

2015-01-01

From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. PMID:26478227
Cell-Free Optogenetic Gene Expression System.

PubMed

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

2018-04-20

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

PubMed

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

2018-04-11

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

PubMed Central

Iyer, Sukanya

2013-01-01

Realizing the potential of cell free systems will require development of ligand sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand sensitive transcriptional control in cell free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single stranded template, results in up to a five-fold change in gene expression in a ligand concentration dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell free systems. PMID:24059754
Cell-Free Expression, Purification, and Characterization of the Functional β2-Adrenergic Receptor for Multianalyte Detection of β-Agonists.

PubMed

Wang, Jian; Liu, Yuan; Zhang, Junhua; Han, Zhengzheng; Wang, Wei; Liu, Yang; Wei, Dong; Huang, Wei

2017-11-01

Large-scale expression of β 2 -adrenergic receptor (β 2 -AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β 2 -AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β 2 -AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β 2 -AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC 50 values were 45.99, 60.38, and 78.02 µg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β 2 -AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.
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.
Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.

PubMed

Zhang, Xu; Lian, Jiazhang; Kai, Lei; Huang, Lei; Cen, Peilin; Xu, Zhinan

2014-02-05

Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹⁴ cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve. Copyright © 2013 Elsevier Inc. All rights reserved.
Cell-free synthetic biology for environmental sensing and remediation.

PubMed

Karig, David K

2017-06-01

The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides tools for transforming these fields through complex integration of natural and novel biological components to achieve sophisticated sensing, regulation, and metabolic function. However, the majority of synthetic biology efforts are conducted in living cells, and concerns over releasing genetically modified organisms constitute a key barrier to environmental applications. Cell-free protein expression systems offer a path towards leveraging synthetic biology, while preventing the spread of engineered organisms in nature. Recent efforts in the areas of cell-free approaches for sensing, regulation, and metabolic pathway implementation, as well as for preserving and deploying cell-free expression components, embody key steps towards realizing the potential of cell-free systems for environmental sensing and remediation. Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.
Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

PubMed

Panthu, Baptiste; Ohlmann, Théophile; Perrier, Johan; Schlattner, Uwe; Jalinot, Pierre; Elena-Herrmann, Bénédicte; Rautureau, Gilles J P

2018-01-19

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.
Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

PubMed

Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

2009-10-01

Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.
Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates.

PubMed

Shrestha, Prashanta; Smith, Mark Thomas; Bundy, Bradley Charles

2014-01-25

Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation. Copyright © 2013 Elsevier B.V. All rights reserved.
Engineering artificial cells by combining HeLa-based cell-free expression and ultra-thin double emulsion template

PubMed Central

Ho, Kwun Yin; Murray, Victoria L.; Liu, Allen P.

2015-01-01

Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors. PMID:25997354
Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin

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

Buehler, Paul W.; Butt, Omer I.; D'Agnillo, Felice, E-mail: felice.dagnillo@fda.hhs.gov

Highlights: {yields} Toxicological implications associated with the use of NaNO{sub 2} therapy to treat systemic cell-free Hb exposure are not well-defined. {yields} Systemic Hb exposure followed by NaNO{sub 2} infusion induces acute CNS toxicities in guinea pigs. {yields} These CNS effects were not reproduced by the infusion of cell-free Hb or NaNO{sub 2} alone. {yields} NaNO{sub 2}-mediated oxidation of cell-free Hb may play a causative role in the observed CNS changes. -- Abstract: Systemic cell-free hemoglobin (Hb) released via hemolysis disrupts vascular homeostasis, in part, through the scavenging of nitric oxide (NO). Sodium nitrite (NaNO{sub 2}) therapy can attenuate themore » hypertensive effects of Hb. However, the chemical reactivity of NaNO{sub 2} with Hb may enhance heme- or iron-mediated toxicities. Here, we investigate the effect of NaNO{sub 2} on the central nervous system (CNS) in guinea pigs exposed to systemic cell-free Hb. Intravascular infusion of NaNO{sub 2}, at doses sufficient to alleviate Hb-mediated blood pressure changes, reduced the expression of occludin, but not zona occludens-1 (ZO-1) or claudin-5, in cerebral tight junctions 4 h after Hb infusion. This was accompanied by increased perivascular heme oxygenase-1 expression, neuronal iron deposition, increased astrocyte and microglial activation, and reduced expression of neuron-specific nuclear protein (NeuN). These CNS changes were not observed in animals treated with Hb or NaNO{sub 2} alone. Taken together, these findings suggest that the use of nitrite salts to treat systemic Hb exposure may promote acute CNS toxicity.« less

Rapid cell-free forward engineering of novel genetic ring oscillators

PubMed Central

Niederholtmeyer, Henrike; Sun, Zachary Z; Hori, Yutaka; Yeung, Enoch; Verpoorte, Amanda; Murray, Richard M; Maerkl, Sebastian J

2015-01-01

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the ‘repressilator’, a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior. DOI: http://dx.doi.org/10.7554/eLife.09771.001 PMID:26430766
Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

NASA Astrophysics Data System (ADS)

Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

2016-02-01

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.
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.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Iyer, Sukanya; Karig, David K; Norred, Sarah E

Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on host E. coli promoters for the construction of circuit elements, such as logic gates, makes implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell free systems. We first present LacImore » repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in a commercially available E. coli cell-free protein expression system. Together, our results contribute to the first demonstration of multi-input regulation of T7 promoters and expand the utility of T7 promoters in cell based as well as cell-free gene circuits.« less
Sealable femtoliter chamber arrays for cell-free biology

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

Retterer, Scott T.; Fowlkes, Jason Davidson; Collier, Charles Patrick

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g. global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) devicemore » contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Lastly, we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques to characterize CFPS gene circuits and their interactions with the cell-free environment.« less
Sealable femtoliter chamber arrays for cell-free biology

DOE PAGES

Retterer, Scott T.; Fowlkes, Jason Davidson; Collier, Charles Patrick; ...

2015-03-11

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g. global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) devicemore » contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Lastly, we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques to characterize CFPS gene circuits and their interactions with the cell-free environment.« less
Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

PubMed

Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.
Cell-free protein synthesis: the state of the art.

PubMed

Whittaker, James W

2013-02-01

Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.
Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses

PubMed Central

Rosenblum, Gabriel; Cooperman, Barry S.

2013-01-01

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression. PMID:24161673
γ-Aminobutyric Acid Type A α4, β2, and δ Subunits Assemble to Produce More Than One Functionally Distinct Receptor Type

PubMed Central

Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry

2014-01-01

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745
Systems, methods and computer readable media for estimating capacity loss in rechargeable electrochemical cells

DOEpatents

Gering, Kevin L.

2013-06-18

A system includes an electrochemical cell, monitoring hardware, and a computing system. The monitoring hardware periodically samples charge characteristics of the electrochemical cell. The computing system periodically determines cell information from the charge characteristics of the electrochemical cell. The computing system also periodically adds a first degradation characteristic from the cell information to a first sigmoid expression, periodically adds a second degradation characteristic from the cell information to a second sigmoid expression and combines the first sigmoid expression and the second sigmoid expression to develop or augment a multiple sigmoid model (MSM) of the electrochemical cell. The MSM may be used to estimate a capacity loss of the electrochemical cell at a desired point in time and analyze other characteristics of the electrochemical cell. The first and second degradation characteristics may be loss of active host sites and loss of free lithium for Li-ion cells.
Rapid discovery of protein interactions by cell-free protein technologies.

PubMed

He, M; Taussig, M J

2007-11-01

Cell-free transcription and translation provides an open, controllable environment for production of correctly folded, soluble proteins and allows the rapid generation of proteins from DNA without the need for cloning. Thus it is becoming an increasingly attractive alternative to conventional in vivo expression systems, especially when parallel expression of multiple proteins is required. Through novel design and exploitation, powerful cell-free technologies of ribosome display and protein in situ arrays have been developed for in vitro production and isolation of protein-binding molecules from large libraries. These technologies can be combined for rapid detection of protein interactions.
Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

PubMed

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

2014-01-01

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

PubMed Central

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

2014-01-01

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

PubMed Central

Butt, Omer I.; Buehler, Paul W.; D'Agnillo, Felice

2011-01-01

Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis. PMID:21356382
Performance benchmarking of four cell-free protein expression systems.

PubMed

Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

2016-02-01

Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.
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.
A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells.

PubMed

Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L; Wetsel, Rick A; Wang, Dachun

2014-02-01

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site-specific targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific Neomycin(R) transgene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of β-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc, and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random integration-free and exogenous reprogramming factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultrastructural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. © 2013 AlphaMed Press.
A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells

PubMed Central

Yan, Qing; Quan, Yuan; Sun, Huanhuan; Peng, Xinmiao; Zou, Zhengyun; Alcorn, Joseph L.; Wetsel, Rick A.; Wang, Dachun

2013-01-01

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector-integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types, are still major obstacles. Here we report a novel strategy using a single non-viral site-specific-targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific NeomycinR trangene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of beta-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random-integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random-integration-free and exogenous reprogramming-factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultra-structural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications. PMID:24123810
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

Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.

PubMed

Kelwick, Richard; Webb, Alexander J; MacDonald, James T; Freemont, Paul S

2016-11-01

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P grac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

NASA Astrophysics Data System (ADS)

Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

2013-12-01

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. Electronic supplementary information (ESI) available: ESI containing 1H NMR spectra and additional fibroblast characterization data. See DOI: 10.1039/c3nr04794f
Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

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

Lewis, Daniel D.; Department of Biomedical Engineering, University of California Davis, Davis, CA; Villarreal, Fernando D.

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with amore » special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.« less
Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

PubMed Central

Lewis, Daniel D.; Villarreal, Fernando D.; Wu, Fan; Tan, Cheemeng

2014-01-01

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems. PMID:25538941
Synthetic biology outside the cell: linking computational tools to cell-free systems.

PubMed

Lewis, Daniel D; Villarreal, Fernando D; Wu, Fan; Tan, Cheemeng

2014-01-01

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.
Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.

PubMed

Zichel, R; Mimran, A; Keren, A; Barnea, A; Steinberger-Levy, I; Marcus, D; Turgeman, A; Reuveny, S

2010-05-01

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.
Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.

PubMed

Makeyev, E V; Kolb, V A; Spirin, A S

1999-02-12

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.
A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

PubMed

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

2015-05-01

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

PubMed Central

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

2017-01-01

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

PubMed

Kasi, Devi; Catherine, Christy; Lee, Seung-Won; Lee, Kyung-Ho; Kim, Yu Jung; Ro Lee, Myeong; Ju, Jung Won; Kim, Dong-Myung

2017-05-01

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017. © 2017 American Institute of Chemical Engineers.
Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems

PubMed Central

Lü, Jian-Ming; Lin, Peter H; Yao, Qizhi; Chen, Changyi

2010-01-01

Abstract Free radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system. PMID:19754673
Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

PubMed

Rosenbaek, Lena L; Rizzo, Federica; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

2017-08-01

The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na + ) and, indirectly, serum potassium (K + ) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ( 22 Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22 Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K + , the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22 Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion. Copyright © 2017 the American Physiological Society.
Uncovering stem-cell heterogeneity in the microniche with label-free microfluidics

NASA Astrophysics Data System (ADS)

Sohn, Lydia L.

2013-03-01

Better suited for large number of cells from bulk tissue, traditional cell-screening techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, they rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. We have developed a label-free, single-cell analysis microfluidic platform capable of quantifying cell-surface marker expression of functional organ stem cells directly isolated from their micro-anatomical niche. With this platform, we have screened single quiescent muscle stem (satellite) cells derived from single myofibers, and we have uncovered an important heterogeneity in the surface-marker expression of these cells. By sorting the screened cells with our microfluidic device, we have determined what this heterogeneity means in terms of muscle stem-cell functionality. For instance, we show that the levels of beta1-integrin can predict the differentiation capacity of quiescent satellite cells, and in contrast to recent literature, that some CXCR4 + cells are not myogenic. Our results provide the first direct demonstration of a microniche-specific variation in gene expression in stem cells of the same lineage. Overall, our label-free, single-cell analysis and cell-sorting platform could be extended to other systems involving rare-cell subsets. This work was funded by the W. M. Keck Foundation, NIH, and California Institute of Regenerative Medicine
Controlling cell-free metabolism through physiochemical perturbations.

PubMed

Karim, Ashty S; Heggestad, Jacob T; Crowe, Samantha A; Jewett, Michael C

2018-01-01

Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

PubMed Central

Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Biedendieck, Rebekka

2018-01-01

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. PMID:29666238
Cell-free protein synthesis for structure determination by X-ray crystallography.

PubMed

Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

2010-01-01

Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.
Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

PubMed

Tokmakov, Alexander A; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

2015-09-11

Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.
Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

PubMed

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.
Differentiation of human pluripotent stem cells into highly functional classical brown adipocytes.

PubMed

Nishio, Miwako; Saeki, Kumiko

2014-01-01

We describe a detailed method for directed differentiation of human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), into functional classical brown adipocytes (BAs) under serum-free and feeder-free conditions. It is a two-tiered culture system, based on very simple techniques, a floating culture and a subsequent adherent culture. It does not require gene transfer. The entire process can be carried out in about 10 days. The key point is the usage of our special hematopoietic cytokine cocktail. Almost all the differentiated cells express uncoupling protein 1, a BA-selective marker, as determined by immunostaining. The differentiated cells show characteristics of classical BA as assessed by morphology and gene/protein expression. Moreover, the expression of myoblast marker genes is transiently induced during the floating culture step. hESC/hiPSC-derived BAs show significantly higher oxygen consumption rates (OCRs) than white adipocytes generated from human mesenchymal stem cell. They also show responsiveness to adrenergic stimuli, with about twofold upregulation in OCR by β-adrenergic receptor (β-AR) agonist treatments. hESC/hiPSC-derived BAs exert in vivo calorigenic activities in response to β-AR agonist treatments as assessed by thermography. Finally, lipid and glucose metabolisms are significantly improved in hESC/hiPSC-derived BA-transplanted mice. Our system provides a highly feasible way to produce functional classical BA bearing metabolism-improving capacities from hESC/hiPSC under a feeder-free and serum-free condition without gene transfer. © 2014 Elsevier Inc. All rights reserved.
Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

PubMed Central

Thoring, Lena; Wüstenhagen, Doreen A.; Borowiak, Maria; Stech, Marlitt; Sonnabend, Andrei; Kubick, Stefan

2016-01-01

Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA. PMID:27684475

A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.

PubMed

Chen, D; Qu, Y; Hua, X; Zhang, L; Liu, Z; Pflugfelder, S C; Li, D-Q

2017-06-01

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.
A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

PubMed

Gehre, Nadine; Nusser, Anja; von Muenchow, Lilly; Tussiwand, Roxane; Engdahl, Corinne; Capoferri, Giuseppina; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

2015-03-01

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Production of G protein-coupled receptors in an insect-based cell-free system.

PubMed

Sonnabend, Andrei; Spahn, Viola; Stech, Marlitt; Zemella, Anne; Stein, Christoph; Kubick, Stefan

2017-10-01

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein-coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell-free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell-free system, performed within a short time and in a cost-effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect-based cell-free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell-free synthesized MOR in comparison to MOR expressed in a human cell line by "one-point" radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328-2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification*

PubMed Central

Takemori, Nobuaki; Takemori, Ayako; Tanaka, Yuki; Endo, Yaeta; Hurst, Jane L.; Gómez-Baena, Guadalupe; Harman, Victoria M.; Beynon, Robert J.

2017-01-01

A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs. Moreover, it is possible to cosynthesize QconCATs in a highly-multiplexed translation reaction, coexpressing tens or hundreds of QconCATs simultaneously. By obviating bacterial culture and through the gain of high level multiplexing, it is now possible to generate tens of thousands of standard peptides in a matter of weeks, rendering absolute quantification of a complex proteome highly achievable in a reproducible, broadly deployable system. PMID:29055021
Hepatic differentiation of human iPSCs in different 3D models: A comparative study.

PubMed

Meier, Florian; Freyer, Nora; Brzeszczynska, Joanna; Knöspel, Fanny; Armstrong, Lyle; Lako, Majlinda; Greuel, Selina; Damm, Georg; Ludwig-Schwellinger, Eva; Deschl, Ulrich; Ross, James A; Beilmann, Mario; Zeilinger, Katrin

2017-12-01

Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures.
Hepatic differentiation of human iPSCs in different 3D models: A comparative study

PubMed Central

Brzeszczynska, Joanna; Knöspel, Fanny; Armstrong, Lyle; Lako, Majlinda; Greuel, Selina; Damm, Georg; Ludwig-Schwellinger, Eva; Deschl, Ulrich; Ross, James A.

2017-01-01

Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures. PMID:29039463
Deep Learning in Label-free Cell Classification

PubMed Central

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-01-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219
Deep Learning in Label-free Cell Classification

NASA Astrophysics Data System (ADS)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-03-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.
Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system.

PubMed

Huang, Edwin P; Marquis, Christopher P; Gray, Peter P

2004-11-20

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (c) 2004 Wiley Periodicals, Inc
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.
Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

PubMed

Kawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, Tohru

2016-06-17

N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.
Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors.

PubMed

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J

2017-03-28

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.
Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Non-Internalizing Receptors

PubMed Central

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J.

2017-01-01

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ~3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or non-internalizing receptor (CD45). When lymphocytes were pre-loaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell anti-tumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors. PMID:28231431
Free radical release and HSP70 expression in two human immune-relevant cell lines after exposure to 1800 MHz radiofrequency radiation.

PubMed

Lantow, M; Schuderer, J; Hartwig, C; Simkó, M

2006-01-01

The goal of this study was to investigate whether radiofrequency (RF) electromagnetic-field (EMF) exposure at 1800 MHz causes production of free radicals and/or expression of heat-shock proteins (HSP70) in human immune-relevant cell systems. Human Mono Mac 6 and K562 cells were used to examine free radical release after exposure to incubator control, sham, RF EMFs, PMA, LPS, heat (40 degrees C) or co-exposure conditions. Several signals were used: continuous-wave, several typical modulations of the Global System for Mobile Communications (GSM): GSM-non DTX (speaking only), GSM-DTX (hearing only), GSM-Talk (34% speaking and 66% hearing) at specific absorption rates (SARs) of 0.5, 1.0, 1.5 and 2.0 W/kg. Heat and PMA treatment induced a significant increase in superoxide radical anions and in ROS production in the Mono Mac 6 cells when compared to sham and/or incubator conditions. No significant differences in free radical production were detected after RF EMF exposure or in the respective controls, and no additional effects on superoxide radical anion production were detected after co-exposure to RF EMFs+PMA or RF EMFs+LPS. The GSM-DTX signal at 2 W/kg produced a significant difference in free radical production when the data were compared to sham because of the decreasing sham value. This difference disappeared when data were compared to the incubator controls. To determine the involvement of heat-shock proteins as a possible inhibitor of free radical production, we investigated the HSP70 expression level after different RF EMF exposures; no significant effects were detected.
Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

PubMed

Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

2014-08-25

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

PubMed Central

Wang, Ying; Hougaard, Anni B.; Paulander, Wilhelm; Skibsted, Leif H.

2015-01-01

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals. PMID:26150471
Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures.

PubMed

Wang, Ying; Hougaard, Anni B; Paulander, Wilhelm; Skibsted, Leif H; Ingmer, Hanne; Andersen, Mogens L

2015-09-01

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Revisiting the putative role of heme as a trigger of inflammation.

PubMed

Vallelian, Florence; Schaer, Christian A; Deuel, Jeremy W; Ingoglia, Giada; Humar, Rok; Buehler, Paul W; Schaer, Dominik J

2018-04-01

Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell-free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb-based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein-binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein-free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein-associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein-associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein-free culture medium demonstrated a weak capacity of heme-solutions to induce toll-like receptor-(TLR4) dependent TNF-alpha expression in macrophages. Our data suggests that the equilibrium-state of free and protein-associated heme critically determines the proinflammatory capacity of the metallo-porphyrin. Based on these data it appears unlikely that inflammation-promoting equilibrium conditions could ever occur in vivo.
Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.

PubMed

Bleckmann, Maren; Schürig, Margitta; Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

2016-01-01

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.
In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

PubMed

Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

2016-06-21

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.
Cell-Free Synthetic Biology Chassis for Nanocatalytic Photon-to-Hydrogen Conversion

DOE PAGES

Wang, Peng; Chang, Angela Y.; Novosad, Valentyn; ...

2017-06-11

We report on entirely man-made nanobio hybrid fabricated through assembly of cell-free expressed transmembrane proton pump and semiconductor nanoparticles as an efficient nanocatalysis for photocatalytic H 2 evolution. The system produces H 2 at a turnover rate of 239 (μmole protein) -1 h -1 under green and 17742 (μmole protein) -1 h -1 under white light at ambient conditions, in water at neutral pH with methanol as a sacrificial electron donor. Robustness and flexibility of this approach allows for systemic manipulation at nanoparticle-bio interface toward directed evolution of energy transformation materials and artificial systems.
Cell-Free Synthetic Biology Chassis for Nanocatalytic Photon-to-Hydrogen Conversion

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

Wang, Peng; Chang, Angela Y.; Novosad, Valentyn

We report on entirely man-made nanobio hybrid fabricated through assembly of cell-free expressed transmembrane proton pump and semiconductor nanoparticles as an efficient nanocatalysis for photocatalytic H 2 evolution. The system produces H 2 at a turnover rate of 239 (μmole protein) -1 h -1 under green and 17742 (μmole protein) -1 h -1 under white light at ambient conditions, in water at neutral pH with methanol as a sacrificial electron donor. Robustness and flexibility of this approach allows for systemic manipulation at nanoparticle-bio interface toward directed evolution of energy transformation materials and artificial systems.
Deep Learning in Label-free Cell Classification

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

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less
Deep Learning in Label-free Cell Classification

DOE PAGES

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; ...

2016-03-15

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less
Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

PubMed Central

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells. PMID:26882313
Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture.

PubMed

Zou, Qing; Wu, Mingjun; Zhong, Liwu; Fan, Zhaoxin; Zhang, Bo; Chen, Qiang; Ma, Feng

2016-01-01

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.
Inhibition of selectin binding

DOEpatents

Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Carolyn

1999-10-05

This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.
Monoacylglycerol lipase promotes metastases in nasopharyngeal carcinoma.

PubMed

Hu, Wen-Rong; Lian, Yi-Fan; Peng, Li-Xia; Lei, Jin-Ju; Deng, Cheng-Cheng; Xu, Miao; Feng, Qi-Sheng; Chen, Li-Zhen; Bei, Jin-Xin; Zeng, Yi-Xin

2014-01-01

Monoacylglycerol lipase (MAGL) is a serine hydrolase that hydrolyzes monoacylglycerides into free fatty acids and glycerol. It has recently been found to be involved in cancer progression through the free fatty acid or endocannabinoid network after studies on its function in the endocannabinoid system. Here, we determined a role for MAGL in nasopharyngeal carcinoma (NPC), which is known for its high metastatic potential. Among the different NPC cells we tested, MAGL was highly expressed in high metastatic NPC cells, whereas low metastatic potential NPC cells exhibited lower expression of MAGL. Overexpression of MAGL in low metastatic NPC cells enhanced their motile behavior and metastatic capacity in vivo. Conversely, knockdown of MAGL reduced the motility of highly metastatic cells, reducing their metastatic capacity in vivo. Growth rate was not influenced by MAGL in either high or low metastatic cells. MAGL expression was associated with the epithelial-mesenchymal transition (EMT) proteins, such as E-cadherin, vimentin and Snail. It was also related to the sidepopulation (SP) of NPC cells. Our findings establish that MAGL promotes metastases in NPC through EMT, and it may serve as a target for the prevention of NPC metastases.
Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

PubMed

Nakajima, Ryota; Takeda, Shizu

2014-01-01

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.
Enzymatic antioxidant system of endotheliocytes.

PubMed

Sharapov, M G; Goncharov, R G; Gordeeva, A E; Novoselov, V I; Antonova, O A; Tikhaze, A K; Lankin, V Z

2016-11-01

It is shown that endothelial cells from human umbilical vein have a reduced activity and gene expression of the "classic" antioxidant enzymes (Cu,Zn-superoxide dismutase, catalase, and Se-containing glutathione peroxidase). At the same time, a high expression level of peroxiredoxin genes was identified in the same endothelial cells, which obviously indicates the predominant involvement of these enzymes in protecting the endothelium from the damaging effect of free radical peroxidation.
Gene expression analysis of a porcine hepatocyte/bile duct in vitro differentiaion model

USDA-ARS?s Scientific Manuscript database

A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes w...
Label-free enumeration, collection and downstream cytological and cytogenetic analysis of circulating tumor cells.

PubMed

Dhar, Manjima; Pao, Edward; Renier, Corinne; Go, Derek E; Che, James; Montoya, Rosita; Conrad, Rachel; Matsumoto, Melissa; Heirich, Kyra; Triboulet, Melanie; Rao, Jianyu; Jeffrey, Stefanie S; Garon, Edward B; Goldman, Jonathan; Rao, Nagesh P; Kulkarni, Rajan; Sollier-Christen, Elodie; Di Carlo, Dino

2016-10-14

Circulating tumor cells (CTCs) have a great potential as indicators of metastatic disease that may help physicians improve cancer prognostication, treatment and patient outcomes. Heterogeneous marker expression as well as the complexity of current antibody-based isolation and analysis systems highlights the need for alternative methods. In this work, we use a microfluidic Vortex device that can selectively isolate potential tumor cells from blood independent of cell surface expression. This system was adapted to interface with three protein-marker-free analysis techniques: (i) an in-flow automated image processing system to enumerate cells released, (ii) cytological analysis using Papanicolaou (Pap) staining and (iii) fluorescence in situ hybridization (FISH) targeting the ALK rearrangement. In-flow counting enables a rapid assessment of the cancer-associated large circulating cells in a sample within minutes to determine whether standard downstream assays such as cytological and cytogenetic analyses that are more time consuming and costly are warranted. Using our platform integrated with these workflows, we analyzed 32 non-small cell lung cancer (NSCLC) and 22 breast cancer patient samples, yielding 60 to 100% of the cancer patients with a cell count over the healthy threshold, depending on the detection method used: respectively 77.8% for automated, 60-100% for cytology, and 80% for immunostaining based enumeration.
Label-free enumeration, collection and downstream cytological and cytogenetic analysis of circulating tumor cells

PubMed Central

Dhar, Manjima; Pao, Edward; Renier, Corinne; Go, Derek E.; Che, James; Montoya, Rosita; Conrad, Rachel; Matsumoto, Melissa; Heirich, Kyra; Triboulet, Melanie; Rao, Jianyu; Jeffrey, Stefanie S.; Garon, Edward B.; Goldman, Jonathan; Rao, Nagesh P.; Kulkarni, Rajan; Sollier-Christen, Elodie; Di Carlo, Dino

2016-01-01

Circulating tumor cells (CTCs) have a great potential as indicators of metastatic disease that may help physicians improve cancer prognostication, treatment and patient outcomes. Heterogeneous marker expression as well as the complexity of current antibody-based isolation and analysis systems highlights the need for alternative methods. In this work, we use a microfluidic Vortex device that can selectively isolate potential tumor cells from blood independent of cell surface expression. This system was adapted to interface with three protein-marker-free analysis techniques: (i) an in-flow automated image processing system to enumerate cells released, (ii) cytological analysis using Papanicolaou (Pap) staining and (iii) fluorescence in situ hybridization (FISH) targeting the ALK rearrangement. In-flow counting enables a rapid assessment of the cancer-associated large circulating cells in a sample within minutes to determine whether standard downstream assays such as cytological and cytogenetic analyses that are more time consuming and costly are warranted. Using our platform integrated with these workflows, we analyzed 32 non-small cell lung cancer (NSCLC) and 22 breast cancer patient samples, yielding 60 to 100% of the cancer patients with a cell count over the healthy threshold, depending on the detection method used: respectively 77.8% for automated, 60–100% for cytology, and 80% for immunostaining based enumeration. PMID:27739521
Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.

PubMed

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

2015-09-21

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

PubMed Central

Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

2015-01-01

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671
In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines

PubMed Central

Tomankova, Katerina; Polakova, Katerina; Pizova, Klara; Binder, Svatopluk; Havrdova, Marketa; Kolarova, Mary; Kriegova, Eva; Zapletalova, Jana; Malina, Lukas; Horakova, Jana; Malohlava, Jakub; Kolokithas-Ntoukas, Argiris; Bakandritsos, Aristides; Kolarova, Hana; Zboril, Radek

2015-01-01

One of the promising strategies for improvement of cancer treatment is based on magnetic drug delivery systems, thus avoiding side effects of standard chemotherapies. Superparamagnetic iron oxide (SPIO) nanoparticles have ideal properties to become a targeted magnetic drug delivery contrast probes, named theranostics. We worked with SPIO condensed colloidal nanocrystal clusters (MagAlg) prepared through a new soft biomineralization route in the presence of alginate as the polymeric shell and loaded with doxorubicin (DOX). The aim of this work was to study the in vitro cytotoxicity of these new MagAlg–DOX systems on mouse fibroblast and breast carcinoma cell lines. For proper analysis and understanding of cell behavior after administration of MagAlg–DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized. It was found that the cytotoxic effect of MagAlg–DOX system is delayed compared to free DOX in both cell lines. This was attributed to the different mechanism of internalization of DOX and MagAlg–DOX into the cells, together with the fact that the drug is strongly bound on the drug nanocarriers. We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line. This is a first comprehensive study on the cytotoxic effect of DOX-loaded SPIO compared with free DOX on healthy and cancer cell lines, as well as on the induced changes in gene expression. PMID:25673990
Construction of a Dual-Fluorescence Reporter System to Monitor the Dynamic Progression of Pluripotent Cell Differentiation.

PubMed

Sun, Wu-Sheng; Chun, Ju-Lan; Do, Jeong-Tae; Kim, Dong-Hwan; Ahn, Jin-Seop; Kim, Min-Kyu; Hwang, In-Sul; Kwon, Dae-Jin; Hwang, Seong-Soo; Lee, Jeong-Woong

2016-01-01

Oct4 is a crucial germ line-specific transcription factor expressed in different pluripotent cells and downregulated in the process of differentiation. There are two conserved enhancers, called the distal enhancer (DE) and proximal enhancer (PE), in the 5' upstream regulatory sequences (URSs) of the mouse Oct4 gene, which were demonstrated to control Oct4 expression independently in embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs). We analyzed the URSs of the pig Oct4 and identified two similar enhancers that were highly consistent with the mouse DE and PE. A dual-fluorescence reporter was later constructed by combining a DE-free- Oct4 -promoter-driven EGFP reporter cassette with a PE-free- Oct4 -promoter-driven mCherry reporter cassette. Then, it was tested in a mouse ESC-like cell line (F9) and a mouse EpiSC-like cell line (P19) before it is formally used for pig. As a result, a higher red fluorescence was observed in F9 cells, while green fluorescence was primarily detected in P19 cells. This fluorescence expression pattern in the two cell lines was consistent with that in the early naïve pluripotent state and late primed pluripotent state during differentiation of mouse ESCs. Hence, this reporter system will be a convenient tool for screening out ESC-like naïve pluripotent stem cells from other metastable state cells in a heterogenous population.
The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria, within the context of a tumour stem cell-like phenotype.

PubMed

Ruggeri, Pierdomenico; Farina, Antonietta R; Di Ianni, Natalia; Cappabianca, Lucia; Ragone, Marzia; Ianni, Giulia; Gulino, Alberto; Mackay, Andrew R

2014-01-01

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.
The TrkAIII Oncoprotein Inhibits Mitochondrial Free Radical ROS-Induced Death of SH-SY5Y Neuroblastoma Cells by Augmenting SOD2 Expression and Activity at the Mitochondria, within the Context of a Tumour Stem Cell-like Phenotype

PubMed Central

Di Ianni, Natalia; Cappabianca, Lucia; Ragone, Marzia; Ianni, Giulia; Gulino, Alberto; Mackay, Andrew R.

2014-01-01

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB. PMID:24736663

Development of an ES-like cell culture system (RESC) from rohu, Labeo rohita (Ham.).

PubMed

Goswami, M; Lakra, W S; Yadav, Kamalendra; Jena, J K

2012-12-01

An embryonic stem (ES)-like cell culture system RESC from a commercially important freshwater carp, Labeo rohita, was developed using blastula stage embryos. The cells were cultured in Leibovitz-15 (L-15) medium in gelatin-coated cell culture flask supplemented with 15 % fetal bovine serum along with 10 ng ml(-1) basic fibroblast growth factor at 28 °C under feeder-free conditions. The ES-like cells were characterized by their unique morphology, alkaline phosphatase activity, embryoid body formation tendency, expression of transcription factor Oct4, and consistent chromosome count. The RESC cells when treated with retinoic acid differentiated into cells of different lineages. The RESC developed from mid-blastula embryos of L. rohita would be a useful tool for cellular differentiation and gene expression studies.
From Blood Islands to Blood Vessels: Morphologic Observations and Expression of Key Molecules during Hyaloid Vascular System Development

PubMed Central

McLeod, D. Scott; Hasegawa, Takuya; Baba, Takayuki; Grebe, Rhonda; Galtier d'Auriac, Ines; Merges, Carol; Edwards, Malia; Lutty, Gerard A.

2012-01-01

Purpose. The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. Methods. Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. Results. Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5–7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. Conclusions. Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts. PMID:23092923
An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

PubMed

Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

2014-05-20

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.
Dedifferentiation of Human Primary Thyrocytes into Multilineage Progenitor Cells without Gene Introduction

PubMed Central

Saenko, Vladimir; Suzuki, Masatoshi; Matsuse, Michiko; Ohtsuru, Akira; Kumagai, Atsushi; Uga, Tatsuya; Yano, Hiroshi; Nagayama, Yuji; Yamashita, Shunichi

2011-01-01

While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (∼0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases. PMID:21556376
Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System.

PubMed

Rustad, Mark; Eastlund, Allen; Marshall, Ryan; Jardine, Paul; Noireaux, Vincent

2017-08-17

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), ΦΧ174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.
Cell-Free RNA Content in Peripheral Blood as Potential Biomarkers for Detecting Circulating Tumor Cells in Non-Small Cell Lung Carcinoma

PubMed Central

Yu, Xin-Min; Wu, Yi-Chen; Liu, Xiang; Huang, Xian-Cong; Hou, Xiu-Xiu; Wang, Jiu-Li; Cheng, Xiang-Liu; Mao, Wei-Min; Ling, Zhi-Qiang

2016-01-01

Circulating tumor cells (CTCs) have been implicated in tumor progression and prognosis. Techniques detecting CTCs in the peripheral blood of patients with non-small cell lung carcinoma (NSCLC) may help to identify individuals likely to benefit from early systemic treatment. However, the detection of CTCs with a single marker is challenging, owing to low specificity and sensitivity and due to the heterogeneity and rareness of CTCs. Herein, the probability of cell-free RNA content in the peripheral blood as a potential biomarker for detecting CTCs in cancer patients was investigated. An immunomagnetic enrichment of real-time reverse-transcription PCR (RT-PCR) technology for analysis of CTCs in NSCLC patients was also developed. The mRNA levels of four candidate genes, cytokeratin 7 (CK7), E74-like factor 3 (ELF3), epidermal growth factor receptor (EGFR), and erythropoietin-producing hepatocellular carcinoma receptor B4 (EphB4) that were significantly elevated in tumor tissues and peripheral blood mononuclear cells (PBMCs) were determined. The expression of CK7 and ELF3 in tumor tissues and EGFR in PBMCs was associated with lymph node metastasis (all p < 0.05). The expression of CK7 in PBMCs was correlated with age and EphB4 in PBMCs correlated with histopathological type, respectively (all p < 0.05). The expression of all four genes in tumor tissues and PBMCs was significantly correlated with the clinical stage (all p < 0.01). Survival analysis showed that the patients with enhanced expression of CK7, ELF3, EGFR, and EphB4 mRNA in PBMCs had poorer disease-free survival (DFS) and overall survival (OS) than those without (all p < 0.0001). The present study showed that this alteration of cell-free RNA content in peripheral blood might have clinical ramifications in the diagnosis and treatment of NSCLC patients. PMID:27827952
Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

PubMed Central

Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

2016-01-01

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816
Surprisal analysis characterizes the free energy time course of cancer cells undergoing epithelial-to-mesenchymal transition.

PubMed

Zadran, Sohila; Arumugam, Rameshkumar; Herschman, Harvey; Phelps, Michael E; Levine, R D

2014-09-09

The epithelial-to-mesenchymal transition (EMT) initiates the invasive and metastatic behavior of many epithelial cancers. Mechanisms underlying EMT are not fully known. Surprisal analysis of mRNA time course data from lung and pancreatic cancer cells stimulated to undergo TGF-β1-induced EMT identifies two phenotypes. Examination of the time course for these phenotypes reveals that EMT reprogramming is a multistep process characterized by initiation, maturation, and stabilization stages that correlate with changes in cell metabolism. Surprisal analysis characterizes the free energy time course of the expression levels throughout the transition in terms of two state variables. The landscape of the free energy changes during the EMT for the lung cancer cells shows a stable intermediate state. Existing data suggest this is the previously proposed maturation stage. Using a single-cell ATP assay, we demonstrate that the TGF-β1-induced EMT for lung cancer cells, particularly during the maturation stage, coincides with a metabolic shift resulting in increased cytosolic ATP levels. Surprisal analysis also characterizes the absolute expression levels of the mRNAs and thereby examines the homeostasis of the transcription system during EMT.
Efficient production of biodiesel from waste grease: one-pot esterification and transesterification with tandem lipases.

PubMed

Yan, Jinyong; Li, Aitao; Xu, Yi; Ngo, Thao P N; Phua, Szechao; Li, Zhi

2012-11-01

A novel concept and efficient method for producing biodiesel (FAME) from grease (15-40wt% free fatty acid, FFA) were developed by using tandem lipases for one-pot esterification of FFA and transesterification of triglyceride with methanol in a solvent-free system. Combining immobilized Candida antarctica lipase B (CALB) (Novozyme 435) favoring the esterification and immobilized Thermomyces lanuginosus lipase (TLL) (Lipozyme TLIM) preferring the transesterification at 2:8 (wt/wt) gave FAME in 80% yield, being better than that with Novozyme 435 or Lipozyme TLIM. Recombinant Escherichia coli (Calb/Tll) co-expressing CALB and TLL was engineered as a more efficient tandem-lipases system. Using wet or dry cells (4wt%) gave FAME in 87% or 95% yield, which is much better than that with E. coli cells expressing either CALB or TLL alone. Cells of E. coli (Calb/Tll) were recycled for five times and retained 75% productivity, thus being practical for producing biodiesel from grease. Copyright © 2012 Elsevier Ltd. All rights reserved.
In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298
Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

PubMed

Locker, Morgane; Kellermann, Odile; Boucquey, Marie; Khun, Huot; Huerre, Michel; Poliard, Anne

2004-01-01

The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.
Feeder & basic fibroblast growth factor-free culture of human embryonic stem cells: Role of conditioned medium from immortalized human feeders.

PubMed

Teotia, Pooja; Sharma, Shilpa; Airan, Balram; Mohanty, Sujata

2016-12-01

Human embryonic stem cell (hESC) lines are commonly maintained on inactivated feeder cells, in the medium supplemented with basic fibroblast growth factor (bFGF). However, limited availability of feeder cells in culture, and the high cost of growth factors limit their use in scalable expansion of hESC cultures for clinical application. Here, we describe an efficient and cost-effective feeder and bFGF-free culture of hESCs using conditioned medium (CM) from immortalized feeder cells. KIND-1 hESC cell line was cultured in CM, collected from primary mouse embryonic fibroblast, human foreskin fibroblast (HFF) and immortalized HFF (I-HFF). Pluripotency of KIND-1 hESC cell line was confirmed by expression of genes, proteins and cell surface markers. In culture, these cells retained normal morphology, expressed all cell surface markers, could differentiate to embryoid bodies upon culture in vitro. Furthermore, I-HFF feeder cells without supplementation of bFGF released ample amount of endogenous bFGF to maintain stemness of hESC cells. The study results described the use of CM from immortalized feeder cells as a consistent source and an efficient, inexpensive feeder-free culture system for the maintenance of hESCs. Moreover, it was possible to maintain hESCs without exogenous supplementation of bFGF. Thus, the study could be extended to scalable expansion of hESC cultures for therapeutic purposes.
Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

PubMed

Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh; Jiang, JinJie; Tokar, Erik J; Malone, Christine C; Petrovich, Robert M; Mason, Ronald P

2017-08-01

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H 2 O 2 ) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H 2 O 2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O 2 •- ) and H 2 O 2 in the presence of NADH. Generation of the free radical O 2 •- and H 2 O 2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies. Published by Elsevier B.V.
Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns.

PubMed

Norred, S Elizabeth; Caveney, Patrick M; Chauhan, Gaurav; Collier, Lauren K; Collier, C Patrick; Abel, Steven M; Simpson, Michael L

2018-05-18

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting-the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increase in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA ("spatial noise") that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. These results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.
Tumour-derived alkaline phosphatase regulates tumour growth, epithelial plasticity and disease-free survival in metastatic prostate cancer

PubMed Central

Rao, S R; Snaith, A E; Marino, D; Cheng, X; Lwin, S T; Orriss, I R; Hamdy, F C; Edwards, C M

2017-01-01

Background: Recent evidence suggests that bone-related parameters are the main prognostic factors for overall survival in advanced prostate cancer (PCa), with elevated circulating levels of alkaline phosphatase (ALP) thought to reflect the dysregulated bone formation accompanying distant metastases. We have identified that PCa cells express ALPL, the gene that encodes for tissue nonspecific ALP, and hypothesised that tumour-derived ALPL may contribute to disease progression. Methods: Functional effects of ALPL inhibition were investigated in metastatic PCa cell lines. ALPL gene expression was analysed from published PCa data sets, and correlated with disease-free survival and metastasis. Results: ALPL expression was increased in PCa cells from metastatic sites. A reduction in tumour-derived ALPL expression or ALP activity increased cell death, mesenchymal-to-epithelial transition and reduced migration. Alkaline phosphatase activity was decreased by the EMT repressor Snail. In men with PCa, tumour-derived ALPL correlated with EMT markers, and high ALPL expression was associated with a significant reduction in disease-free survival. Conclusions: Our studies reveal the function of tumour-derived ALPL in regulating cell death and epithelial plasticity, and demonstrate a strong association between ALPL expression in PCa cells and metastasis or disease-free survival, thus identifying tumour-derived ALPL as a major contributor to the pathogenesis of PCa progression. PMID:28006818
A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

PubMed Central

Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

2016-01-01

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741
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.
Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System.

PubMed

Liu, Kuancheng; Hu, Jianming

2018-04-20

The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions. We have recently developed a mammalian cell-free system based on the rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological concentrations and assembles into capsids under near-physiological conditions. This system has already revealed HBc assembly requirements that are not anticipated based on previous assembly systems. Furthermore, capsid assembly in this system is regulated by endogenous host factors that can be readily manipulated. Here we present a detailed protocol for this cell-free capsid assembly system, including an illustration on how to manipulate host factors that regulate assembly.
Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome.

PubMed

Massingham, Lauren J; Johnson, Kirby L; Scholl, Thomas M; Slonim, Donna K; Wick, Heather C; Bianchi, Diana W

2014-09-01

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.
Interactions between rnacrophage cytokines and eicosanoids in expression of antitumour activity

PubMed Central

Ben-Efraim, Shlomo

1992-01-01

Cytokines and eicosanoid products of macrophages play an essential role in expression of antitumour activity of macrophages either in a cell-to-cell contact system between the effector and the target cell or as cell-free soluble products. In this review the relationship between three main monokines, namely TNF-α, IL-1 and IL-6 and the interrelationship between these monokines and eicosanoids (PGE2, PGI2, LTB4, LTC4) in their production and in expression of antitumour activity is discussed. Emphasis is given to the effect of tumour burden on production of the monokines and of the eicosanoids and on the production of these compounds by the tumour cells. Finally, the therapeutic implications drawn from animal studies and clinical trials is discussed. PMID:18475475

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

PubMed Central

Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004
Protein Degradation in a TX-TL Cell-free Expression System Using ClpXP Protease

DTIC Science & Technology

2014-07-14

function in TX-TL, as well as bacteriophage assembly [2, 6]. Circuits can also be prototyped from basic parts within 8 hours, avoiding cloning and...mRFP, and Venus and variants eGFP-ssrA, mRFP-ssrA, and Venus-ssrA, coding sequences were cloned into a T7-lacO inducible vector containing a N...12672L12677.! 6.! Shin,!J.,!P.!Jardine,!and!V.!Noireaux,!Genome(Replication,(Synthesis,(and( Assembly(of(the( Bacteriophage (T7(in(a(Single(Cell9Free
Enhanced cell migration and apoptosis resistance may underlie the association between high SERPINE1 expression and poor outcome in head and neck carcinoma patients

PubMed Central

Téllez-Gabriel, Marta; León, Xavier; Virós, David; López, Montserrat; Gallardo, Alberto; Céspedes, Maria Virtudes; Casanova, Isolda; López-Pousa, Antonio; Mangues, Maria Antonia; Quer, Miquel; Barnadas, Agustí; Mangues, Ramón

2015-01-01

High SERPINE1 expression is a common event in head and neck squamous cell carcinoma (HNSCC); however, whether it plays a role in determining clinical outcome remains still unknown. We studied SERPINE1 as a prognostic marker in two HNSCC patient cohorts. In a retrospective study (n = 80), high expression of SERPINE1 was associated with poor progression-free (p = 0.022) and cancer-specific (p = 0.040) survival. In a prospective study (n = 190), high SERPINE1 expression was associated with poor local recurrence-free (p = 0.022), progression-free (p = 0.002) and cancer-specific (p = 0.006) survival. SERPINE1 expression was identified as an independent risk factor for progression-free survival in patients treated with chemo-radiotherapy or radiotherapy (p = 0.043). In both patient cohorts, high SERPINE1 expression increased the risk of metastasis spread (p = 0.045; p = 0.029). The association between SERPINE1 expression and survival was confirmed using the HNSCC cohort included in The Cancer Genome Atlas project (n = 507). Once again, patients showing high expression had a poorer survival (p < 0.001). SERPINE1 over-expression in HNSCC cells reduced cell proliferation and enhanced migration. It also protected cells from cisplatin-induced apoptosis, which was accompanied by PI3K/AKT pathway activation. Downregulation of SERPINE1 expression had the opposite effect. We propose SERPINE1 expression as a prognostic marker that could be used to stratify HNSCC patients according to their risk of recurrence. PMID:26359694
Development of serum-free media for lepidopteran insect cell lines.

PubMed

Agathos, Spiros N

2007-01-01

Lepidopteran insect cell culture technology has progressed to the point of becoming an essential part of one of the most successful eukaryotic expression systems and is increasingly used industrially on a large scale. Therefore, there is a constant need for convenient and low-cost culture media capable of supporting good insect cell growth and ensuring high yield of baculovirus as well as the strong expression of recombinant proteins. Vertebrate sera or invertebrate hemolymph were essential supplements in first-generation insect cell media. These supplements, however, are cumbersome and expensive for routine large-scale culture; thus, their use is now circumvented by substituting the essential growth factors present in these supplements with serum-free substances. Such non-serum supplements are typically of non-animal origin and include protein hydrolysates, lipid emulsions, and specialized substances (e.g., surfactants and shear damage protecting chemicals). These supplements need to complement the defined, synthetic basal medium to ensure that the fundamental nutritional needs of the cells are satisfied. Although there is a significant number of proprietary serum-free and low-protein or protein-free media on the market, the lack of information concerning their detailed composition is a drawback in their adoption for different applications, including their adaptation to the metabolic and kinetic analysis and monitoring of a given insect cell based bioprocess. Hence, there is wide appeal for formulating serum-free media based on a rational assessment of the metabolic requirements of the lepidopteran cells during both the growth and the production phases. Techniques such as statistical experimental design and genetic algorithms adapted to the cellular behavior and the bioreactor operation mode (batch, fed-batch, or perfusion) permit the formulation of versatile serum- and protein-free media. These techniques are illustrated with recent developments of serum-free media for the cultivation of commercially important Spodoptera frugiperda and Trichoplusia ni cell lines.
Cell-Free Synthetic Biology Chassis for Nanocatalytic Photon-to-Hydrogen Conversion.

PubMed

Wang, Peng; Chang, Angela Y; Novosad, Valentyn; Chupin, Vladimir V; Schaller, Richard D; Rozhkova, Elena A

2017-07-25

We report on an entirely man-made nano-bio architecture fabricated through noncovalent assembly of a cell-free expressed transmembrane proton pump and TiO 2 semiconductor nanoparticles as an efficient nanophotocatalyst for H 2 evolution. The system produces hydrogen at a turnover of about 240 μmol of H 2 (μmol protein) -1 h -1 and 17.74 mmol of H 2 (μmol protein) -1 h -1 under monochromatic green and white light, respectively, at ambient conditions, in water at neutral pH and room temperature, with methanol as a sacrificial electron donor. Robustness and flexibility of this approach allow for systemic manipulation at the nanoparticle-bio interface toward directed evolution of energy transformation materials and artificial systems.
Glutathione reductase mediates drug resistance in glioblastoma cells by regulating redox homeostasis.

PubMed

Zhu, Zhongling; Du, Shuangshuang; Du, Yibo; Ren, Jing; Ying, Guoguang; Yan, Zhao

2018-01-01

Glutathione (GSH) and GSH-related enzymes constitute the most important defense system that protects cells from free radical, radiotherapy, and chemotherapy attacks. In this study, we aim to explore the potential role and regulatory mechanism of the GSH redox cycle in drug resistance in glioblastoma multiforme (GBM) cells. We found that temozolomide (TMZ)-resistant glioma cells displayed lower levels of endogenous reactive oxygen species and higher levels of total antioxidant capacity and GSH than sensitive cells. Moreover, the expression of glutathione reductase (GSR), the key enzyme of the GSH redox cycle, was higher in TMZ-resistant cells than in sensitive cells. Furthermore, silencing GSR in drug-resistant cells improved the sensitivity of cells to TMZ or cisplatin. Conversely, the over-expression of GSR in sensitive cells resulted in resistance to chemotherapy. In addition, the GSR enzyme partially prevented the oxidative stress caused by pro-oxidant L-buthionine -sulfoximine. The modulation of redox state by GSH or L-buthionine -sulfoximine regulated GSR-mediated drug resistance, suggesting that the action of GSR in drug resistance is associated with the modulation of redox homeostasis. Intriguingly, a trend toward shorter progress-free survival was observed among GBM patients with high GSR expression. These results indicated that GSR is involved in mediating drug resistance and is a potential target for improving GBM treatment. © 2017 International Society for Neurochemistry.
CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

PubMed

Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

2016-07-15

CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.
Whole-cell based solvent-free system for one-pot production of biodiesel from waste grease.

PubMed

Li, Aitao; Ngo, Thao P N; Yan, Jinyong; Tian, Kaiyuan; Li, Zhi

2012-06-01

A whole-cell based solvent-free system was developed for efficient conversion of waste grease to biodiesel via one-pot esterification and transesterification. By isolation and screening of lipase-producing strains from soil, Serratia marcescens YXJ-1002 was discovered for the biotransformation of grease to biodiesel. The lipase (SML) from this strain was cloned and expressed in Escherichia coli as an intracellular enzyme, showing 6 times higher whole-cell based hydrolysis activity than that of wild type strain. The recombinant cells were used for biodiesel production from waste grease in one-pot reactions containing no solvent with the addition of methanol in several small portions, and 97% yield of biodiesel (FAME) was achieved under optimized conditions. In addition, the whole-cell biocatalysts showed excellent reusability, retaining 74% productivity after 4 cycles. The developed system, biocatalyst, and process enable the efficient, low-cost, and green production of biodiesel from waste grease, providing with a potential industrial application. Copyright © 2012 Elsevier Ltd. All rights reserved.
Mechanisms of foam cell formation in atherosclerosis.

PubMed

Chistiakov, Dimitry A; Melnichenko, Alexandra A; Myasoedova, Veronika A; Grechko, Andrey V; Orekhov, Alexander N

2017-11-01

Low-density lipoprotein (LDL) and cholesterol homeostasis in the peripheral blood is maintained by specialized cells, such as macrophages. Macrophages express a variety of scavenger receptors (SR) that interact with lipoproteins, including SR-A1, CD36, and lectin-like oxLDL receptor-1 (LOX-1). These cells also have several cholesterol transporters, including ATP-binding cassette transporter ABCA1, ABCG1, and SR-BI, that are involved in reverse cholesterol transport. Lipids internalized by phagocytosis are transported to late endosomes/lysosomes, where lysosomal acid lipase (LAL) digests cholesteryl esters releasing free cholesterol. Free cholesterol in turn is processed by acetyl-CoA acetyltransferase (ACAT1), an enzyme that transforms cholesterol to cholesteryl esters. The endoplasmic reticulum serves as a depot for maintaining newly synthesized cholesteryl esters that can be processed by neutral cholesterol ester hydrolase (NCEH), which generates free cholesterol that can exit via cholesterol transporters. In atherosclerosis, pro-inflammatory stimuli upregulate expression of scavenger receptors, especially LOX-1, and downregulate expression of cholesterol transporters. ACAT1 is also increased, while NCEH expression is reduced. This results in deposition of free and esterified cholesterol in macrophages and generation of foam cells. Moreover, other cell types, such as endothelial (ECs) and vascular smooth muscle cells (VSMCs), can also become foam cells. In this review, we discuss known pathways of foam cell formation in atherosclerosis.
Release of cell-free ice nuclei from Halomonas elongata expressing the ice nucleation gene inaZ of Pseudomonas syringae.

PubMed

Tegos, G; Vargas, C; Perysinakis, A; Koukkou, A I; Christogianni, A; Nieto, J J; Ventosa, A; Drainas, C

2000-11-01

Release of ice nuclei in the growth medium of recombinant Halomonas elongata cells expressing the inaZ gene of Pseudomonas syringae was studied in an attempt to produce cell-free active ice nuclei for biotechnological applications. Cell-free ice nuclei were not retained by cellulose acetate filters of 0.2 microm pore size. Highest activity of cell-free ice nuclei was obtained when cells were grown in low salinity (0.5-5% NaCl, w/v). Freezing temperature threshold, estimated to be below -7 degrees C indicating class C nuclei, was not affected by medium salinity. Their density, as estimated by Percoll density centrifugation, was 1.018 +/- 0.002 gml(-1) and they were found to be free of lipids. Ice nuclei are released in the growth medium of recombinant H. elongata cells probably because of inefficient anchoring of the ice-nucleation protein aggregates in the outer membrane. The ice+ recombinant H. elongata cells could be useful for future use as a source of active cell-free ice nucleation protein.
Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent

PubMed Central

van Erp, Anke E.M.; Versleijen-Jonkers, Yvonne M.H.; Hillebrandt-Roeffen, Melissa H.S.; van Houdt, Laurens; Gorris, Mark A.J.; van Dam, Laura S.; Mentzel, Thomas; Weidema, Marije E.; Savci-Heijink, C. Dilara; Desar, Ingrid M.E.; Merks, Hans H.M.; van Noesel, Max M.; Shipley, Janet; van der Graaf, Winette T.A.; Flucke, Uta E.; Meyer-Wentrup, Friederike A.G.

2017-01-01

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma (n = 46), Ewing sarcoma (n = 32), alveolar rhabdomyosarcoma (n = 20), embryonal rhabdomyosarcoma (n = 77), synovial sarcoma (n = 22) and desmoplastic small round cell tumors (DSRCT) (n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described. PMID:29050367
Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent.

PubMed

van Erp, Anke E M; Versleijen-Jonkers, Yvonne M H; Hillebrandt-Roeffen, Melissa H S; van Houdt, Laurens; Gorris, Mark A J; van Dam, Laura S; Mentzel, Thomas; Weidema, Marije E; Savci-Heijink, C Dilara; Desar, Ingrid M E; Merks, Hans H M; van Noesel, Max M; Shipley, Janet; van der Graaf, Winette T A; Flucke, Uta E; Meyer-Wentrup, Friederike A G

2017-09-19

In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma ( n = 46), Ewing sarcoma ( n = 32), alveolar rhabdomyosarcoma ( n = 20), embryonal rhabdomyosarcoma ( n = 77), synovial sarcoma ( n = 22) and desmoplastic small round cell tumors (DSRCT) ( n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described.
Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR.

PubMed

Nomoto, Mika; Tada, Yasuomi

2018-01-01

Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.
Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns

DOE PAGES

Norred, Sarah Elizabeth; Caveney, Patrick M.; Chauhan, Gaurav; ...

2018-04-24

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting—the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increasemore » in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA (“spatial noise”) that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. Furthermore, these results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.« less
Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns

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

Norred, Sarah Elizabeth; Caveney, Patrick M.; Chauhan, Gaurav

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting—the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increasemore » in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA (“spatial noise”) that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. Furthermore, these results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.« less
Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

PubMed

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

2014-05-03

Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.
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.
N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

PubMed

Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

2010-07-01

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.
Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

PubMed

Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, Maria J; Romero, Fernando; Gil, Angel

2013-01-01

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro-inflammatory pathways.
Cell-Free Culture Supernatant of Bifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged with Salmonella typhi through TLR Activation

PubMed Central

Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, Maria J.; Romero, Fernando; Gil, Angel

2013-01-01

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro-inflammatory pathways. PMID:23555025

Molecular characterization and expression profile of nanos in Schistosoma japonicum and its influence on the expression several mammalian stem cell factors.

PubMed

Giri, Bikash Ranjan; Du, Xiaoli; Xia, Tianqi; Chen, Yongjun; Li, Hao; Cheng, Guofeng

2017-07-01

Pluripotent stem cells, called neoblasts, are well known for the regenerative capability and developmental plasticity in flatworms. Impressive advancement has been made in free-living flatworms, while in case of its parasitic counterpart, neoblast-like stem cells have attracted recent attention for its self-renewal and differentiation capacity. Nanos is a key conserved post-transcriptional regulator critical for the formation, development, and/or maintenance of the pluripotent germ line stem cell systems in many metazoans including schistosomes. In the present study, we report the molecular cloning and expression of nanos orthologous genes nanos in Schistosoma japonicum (Sjnanos). The cDNA of Sjnanos is 826 bp long, containing an open reading frame (ORF) for 223 amino acid long protein. qRT-PCR analysis shown that Sjnanos was differently expressed in several stages of schistosomes with relatively high level in schistosomula. Additionally, Sjnanos was expressed highly in adult females compared to adult males. Transfection of recombinant plasmid for expressing Sjnanos resulted in significant proliferation and increased expression of several stem cell factors in mammalian cells. Overall, our preliminary study provides the molecular basis to further functionally characterize Sjnanos in S. japonicum.
Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

PubMed

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.
Housing system influences abundance of Pax3 and Pax7 in postnatal chicken skeletal muscles.

PubMed

Yin, H D; Li, D Y; Zhang, L; Yang, M Y; Zhao, X L; Wang, Y; Liu, Y P; Zhu, Q

2014-06-01

Paired box (Pax) proteins 3 and 7 are associated with activation of muscle satellite cells and play a major role in hyperplastic and hypertrophic growth in postnatal skeletal muscle fibers. The objective of this study was to evaluate the effect of housing system on abundance of Pax3 and Pax7 in postnatal chicken skeletal muscles. At 42 d, 1,200 chickens with similar BW were randomly assigned to cage, pen, and free-range group. The mRNA abundance was measured in pectoralis major and thigh muscle at d 56, 70, and 84, and the protein expression was quantified at d 84. Increases in mRNA abundance of PAX3 and PAX7 with age were less pronounced in caged system chickens than in pen and free-range chickens from d 56 to 84, and free-range chickens showed a more pronounced increase in gene expression with age compared with penned chickens. At d 84, quantities of PAX3 and PAX7 mRNA and protein were highest in both pectoralis major and thigh muscle of chickens raised in the free-range group, lowest in penned chickens, and intermediate in caged chickens (P < 0.05). These data indicate that housing system may influence muscle fiber muscle accretion by coordinating the expression of Pax3 and Pax7 in adult chicken skeletal muscles. Poultry Science Association Inc.
Expression of membrane-associated proteins within single emulsion cell facsimiles.

PubMed

Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

2012-07-07

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.
Phenotyping and comparing the immune cell populations of free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) and dolphins under human care.

PubMed

Nouri-Shirazi, Mahyar; Bible, Brittany F; Zeng, Menghua; Tamjidi, Saba; Bossart, Gregory D

2017-03-27

Studies suggest that free-ranging bottlenose dolphins exhibit a suppressed immune system because of exposure to contaminants or microorganisms. However, due to a lack of commercially available antibodies specific to marine mammal immune cell surface markers, the research has been indecisive. The purpose of this study was to identify cross-reactive terrestrial-specific antibodies in order to assess the changes in the immune cell populations of dolphins under human care and free-ranging dolphins. The blood and PBMC fraction of blood samples from human care and free-ranging dolphins were characterized by H&E staining of cytospin slides and flow cytometry using a panel of terrestrial-specific antibodies. In this study, we show that out of 65 terrestrial-specific antibodies tested, 11 were cross-reactive and identified dolphin immune cell populations within their peripheral blood. Using these antibodies, we found significant differences in the absolute number of cells expressing specific markers within their lymphocyte and monocyte fractions. Interestingly, the peripheral blood mononuclear cell profile of free-ranging dolphins retained an additional population of cells that divided them into two groups showing a low (<27%) or high (>56%) percentage of smaller cells resembling granulocytes. We found that the cross-reactive antibodies not only identified specific changes in the immune cells of free-ranging dolphins, but also opened the possibility to investigate the causal relationship between immunosuppression and mortality seen in free-ranging dolphins.
Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

NASA Astrophysics Data System (ADS)

Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

2010-05-01

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.
Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

PubMed Central

Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

2015-01-01

Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101
Preservation of protein expression systems at elevated temperatures for portable therapeutic production

PubMed Central

Bessling, Seneca; Thielen, Peter; Zhang, Sherry; Wolfe, Joshua

2017-01-01

Many biotechnology capabilities are limited by stringent storage needs of reagents, largely prohibiting use outside of specialized laboratories. Focusing on a large class of protein-based biotechnology applications, we address this issue by developing a method for preserving cell-free protein expression systems for months above room temperature. Our approach realizes unprecedented long-term stability at elevated temperatures by leveraging the sugar alcohol trehalose, a simple, low-cost, open-air drying step, and strategic separation of reaction components during drying. The resulting preservation capacity enables efficient production of a wide range of on-demand proteins under adverse conditions, for instance during emergency outbreaks or in remote locations. To demonstrate application potential, we use cell-free reagents subjected to months of exposure at 37°C and atmospheric conditions to produce sufficient concentrations of a pyocin protein to kill Pseudomonas aeruginosa, a troublesome pathogen for traumatic and burn wound injuries. Our work makes possible new biotechnology applications that demand ruggedness and scalability. PMID:28446704
Label-Free Raman Microspectral Analysis for Comparison of Cellular Uptake and Distribution between Non-Targeted and EGFR-Targeted Biodegradable Polymeric Nanoparticles

PubMed Central

Chernenko, Tatyana; Buyukozturk, Fulden; Miljkovic, Milos; Carrier, Rebecca; Diem, Max; Amiji, Mansoor

2013-01-01

Active targeted delivery of nanoparticle-encapsulated agents to tumor cells in vivo is expected to enhance therapeutic effect with significantly less non-specific toxicity. Active targeting is based on surface modification of nanoparticles with ligands that bind with extracellular targets and enhance payload delivery in the cells. In this study, we have used label-free Raman micro-spectral analysis and kinetic modeling to study cellular interactions and intracellular delivery of C6-ceramide using a non-targeted and an epidermal growth factor receptor (EGFR) targeted biodegradable polymeric nano-delivery systems, in EGFR-expressing human ovarian adenocarcinoma (SKOV3) cells. The results show that EGFR peptide-modified nanoparticles were rapidly internalized in SKOV3 cells leading to significant intracellular accumulation as compared to non-specific uptake by the non-targeted nanoparticles. Raman micro-spectral analysis enables visualization and quantification of the carrier system, drug-load, and responses of the biological systems interrogated, without exogenous staining and labeling procedures. PMID:24298430
Molecular Ghrelin System in the Pancreatic Acinar Cells: The Role of the Polypeptide, Caerulein and Sensory Nerves.

PubMed

Bonior, Joanna; Ceranowicz, Piotr; Gajdosz, Ryszard; Kuśnierz-Cabala, Beata; Pierzchalski, Piotr; Warzecha, Zygmunt; Dembiński, Artur; Pędziwiatr, Michał; Kot, Michalina; Leja-Szpak, Anna; Nawrot-Porąbka, Katarzyna; Link-Lenczowski, Paweł; Olszanecki, Rafał; Bartuś, Krzysztof; Jaworek, Jolanta

2017-05-02

Ghrelin (GHRL) is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Experimental studies showed that GHRL protects the stomach and pancreas against acute damage, but the effect of GHRL on pancreatic acinar cells was still undetermined. To investigate the effect of GHRL and caerulein on the functional ghrelin system in pancreatic acinar cells taking into account the role of sensory nerves (SN). Experiments were carried out on isolated pancreatic acinar cells and AR42J cells. Before acinar cells isolation, GHRL was administered intraperitoneally at a dose of 50 µg/kg to rats with intact SN or with capsaicin deactivation of SN (CDSN). After isolation, pancreatic acinar cells were incubated in caerulein-free or caerulein containing solution. AR42J cells were incubated under basal conditions and stimulated with caerulein, GHRL or a combination of the above. Incubation of isolated acinar cells with caerulein inhibited GHS-R and GHRL expression at the level of mRNA and protein in those cells. Either in rats with intact SN or with CDSN, administration of GHRL before isolation of acinar cells increased expression of GHRL and GHS-R in those cells and reversed the caerulein-induced reduction in expression of those parameters. Similar upregulation of GHS-R and GHRL was observed after administration of GHRL in AR42J cells. GHRL stimulates its own expression and expression of its receptor in isolated pancreatic acinar cells and AR42J cells on the positive feedback pathway. This mechanism seems to participate in the pancreatoprotective effect of GHRL in the course of acute pancreatitis.
Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

DOE PAGES

He, Wei; Felderman, Martina; Evans, Angela C.; ...

2017-07-24

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, andmore » protein misfolding. For this study, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP–tNLP). The cell-free expressed mMOMP–tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP–tNLP complex in a 1-ml cell-free reaction. The mMOMP–tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP–tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.« less
Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

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

He, Wei; Felderman, Martina; Evans, Angela C.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, andmore » protein misfolding. For this study, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP–tNLP). The cell-free expressed mMOMP–tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP–tNLP complex in a 1-ml cell-free reaction. The mMOMP–tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP–tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.« less
Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302.

PubMed

Nishimura, Ken; Ohtaka, Manami; Takada, Hitomi; Kurisaki, Akira; Tran, Nhi Vo Kieu; Tran, Yen Thi Hai; Hisatake, Koji; Sano, Masayuki; Nakanishi, Mahito

2017-08-01

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3' untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes.

PubMed

Kim, Hye Kyung

2016-07-29

Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes.
Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes

PubMed Central

Kim, Hye Kyung

2016-01-01

Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes. PMID:27483310
Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

PubMed

Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.
Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

PubMed Central

Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412
Expression regulation by a methyl-CpG binding domain in an E. coli based, cell-free TX-TL system

NASA Astrophysics Data System (ADS)

Schenkelberger, M.; Shanak, S.; Finkler, M.; Worst, E. G.; Noireaux, V.; Helms, V.; Ott, A.

2017-04-01

Cytosine methylation plays an important role in the epigenetic regulation of eukaryotic gene expression. The methyl-CpG binding domain (MBD) is common to a family of eukaryotic transcriptional regulators. How MBD, a stretch of about 80 amino acids, recognizes CpGs in a methylation dependent manner, and as a function of sequence, is only partly understood. Here we show, using an Escherichia coli cell-free expression system, that MBD from the human transcriptional regulator MeCP2 performs as a specific, methylation-dependent repressor in conjunction with the BDNF (brain-derived neurotrophic factor) promoter sequence. Mutation of either base flanking the central CpG pair changes the expression level of the target gene. However, the relative degree of repression as a function of MBD concentration remains unaltered. Molecular dynamics simulations that address the DNA B fiber ratio and the handedness reveal cooperative transitions in the promoter DNA upon MBD binding that correlate well with our experimental observations. We suggest that not only steric hindrance, but also conformational changes of the BDNF promoter as a result of MBD binding are required for MBD to act as a specific inhibitory element. Our work demonstrates that the prokaryotic transcription machinery can reproduce features of epigenetic mammalian transcriptional regulatory elements.
Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells.

PubMed

Winkler, Monica E; Mauritz, Christina; Groos, Stephanie; Kispert, Andreas; Menke, Sandra; Hoffmann, Anika; Gruh, Ina; Schwanke, Kristin; Haverich, Axel; Martin, Ulrich

2008-03-01

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.
Analysis of the cytochrome c-dependent apoptosis apparatus in cells from human pancreatic carcinoma

PubMed Central

Gerhard, M C; Schmid, R M; Häcker, G

2002-01-01

Defects in the apoptotic system are likely to play a role in tumorigenesis. Pancreatic carcinoma cells are extremely resistant to apoptosis induction by chemotherapy suggesting that the apoptosis machinery is faulty. We investigated the integrity of the cytochrome c-dependent apoptotic apparatus in 10 human pancreatic carcinoma cell lines. Expression of Apaf-1, caspase-3, -6, -7, -8 and -9, Hsp-70 and XIAP was detected in all cell lines. The expression levels of Apaf-1 and caspase-8 were homogenous in all cell lines whereas differences in expression of other caspases were seen. In cytosolic fractions, all investigated caspases were processed in response to cytochrome c but the extent of processing varied between the cell lines. No stringent correlation between the amount of processing of caspase-9 and effector caspases was seen. Cytochrome c-induced effector caspase activity was quantitated by enzyme assay. Especially at low concentrations of added cytochrome c, this response varied greatly between the cell lines. These data demonstrate that the apoptotic system downstream of the mitochondria is qualitatively intact in pancreatic carcinoma. They further show that the response to cytochrome c can be quantitated in a cell-free system and that determinants other than mere expression of apoptotic molecules can regulate cytochrome c-induced apoptosis. British Journal of Cancer (2002) 86, 893–898. DOI: 10.1038/sj/bjc/6600171 www.bjcancer.com © 2002 Cancer Research UK PMID:11953820

High yield cell-free production of integral membrane proteins without refolding or detergents.

PubMed

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.
Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

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

Cleveland, Thomas E.; He, Wei; Evans, Angela C.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less
Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

DOE PAGES

Cleveland, Thomas E.; He, Wei; Evans, Angela C.; ...

2018-02-13

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less
YB-1, the E2F Pathway, and Regulation of Tumor Cell Growth

PubMed Central

Samuel, Weini; Cao, Helen; Patel, Rachna; Mehta, Reena; Stern, J. Lewis; Reid, Glen; Woolley, Adele G.; Miller, Lance D.; Black, Michael A; Shelling, Andrew N.; Print, Cristin G.; Braithwaite, Antony W.

2012-01-01

Background Y-box binding factor 1 (YB-1) has been associated with prognosis in many tumor types. Reduced YB-1 expression inhibits tumor cell growth, but the mechanism is unclear. Methods YB-1 mRNA levels were compared with tumor grade and histology using microarray data from 771 breast cancer patients and with disease-free survival and distant metastasis–free survival using data from 375 of those patients who did not receive adjuvant therapy. Microarrays were further searched for genes that had correlated expression with YB-1 mRNA. Small interfering RNA (siRNA) was used to study the effects of reduced YB-1 expression on growth of three tumor cell lines (MCF-7 breast, HCT116 colon, and A549 lung cancer cells), on tumorigenesis by A549 cells in nude mice, and on global transcription in the three cancer cell lines. Reporter gene assays were used to determine whether YB-1 siRNAs affected the expression of E2F1, and chromatin immunoprecipitation was used to determine whether YB-1 bound to various E2F promoters as well as E2F1-regulated promoters. All P values were from two-sided tests. Results YB-1 levels were elevated in more aggressive tumors and were strongly associated with poor disease-free survival and distant metastasis–free survival. YB-1 expression was often associated with the expression of genes with E2F sites in their promoters. Cells expressing YB-1 siRNA grew substantially more slowly than control cells and formed tumors less readily in nude mice. Transcripts that were altered in cancer cell lines with YB-1 siRNA included 32 genes that are components of prognostic gene expression signatures. YB-1 regulated expression of an E2F1 promoter–reporter construct in A549 cells (eg, relative E2F1 promoter activity with control siRNA = 4.04; with YB-1 siRNA = 1.40, difference= −2.64, 95% confidence interval = −3.57 to −1.71, P < .001) and bound to the promoters of several well-defined E2F1 target genes. Conclusion YB-1 expression is associated with the activity of E2F transcription factors and may control tumor cell growth by this mechanism. PMID:22205655
Tissue plasminogen activator (tPA) as a reporter gene in transient gene expression.

PubMed

Cheng, S M; Lee, S G; Kalyan, N K; McCloud, S; Levner, M; Hung, P P

1987-01-01

Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.
Establishing a high yielding streptomyces-based cell-free protein synthesis system.

PubMed

Li, Jian; Wang, He; Kwon, Yong-Chan; Jewett, Michael C

2017-06-01

Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
The potential role of free chitosan in bone trauma and bone cancer management.

PubMed

Tan, Mei L; Shao, Peng; Friedhuber, Anna M; van Moorst, Mallory; Elahy, Mina; Indumathy, Sivanjah; Dunstan, Dave E; Wei, Yongzhong; Dass, Crispin R

2014-09-01

Bone defects caused by fractures or cancer-mediated destruction are debilitating. Chitosan is commonly used in scaffold matrices for bone healing, but rarely as a free drug. We demonstrate that free chitosan promotes osteoblast proliferation and osteogenesis in mesenchymal stem cells, increases osteopontin and collagen I expression, and reduces osteoclastogenesis. Chitosan inhibits invasion of endothelial cells, downregulating uPA/R, MT1-MMP, cdc42 and Rac1. Better healing of bone fractures with greater trabecular bone formation was observed in mice treated with chitosan. Chitosan induces apoptosis in osteotropic prostate and breast cancer cells via caspase-2 and -3 activation, and reduces their establishment in bone. Chitosan is pro-apoptotic in osteosarcoma cells, but not their normal counterpart, osteoblasts, or chondrosarcoma cells. Systemic delivery of chitosan does not perturb angiogenesis, bone volume or instinctive behaviour in pregnant mice, but decreases foetal length and changes pancreatic secretory acini. With certain controls in place, chitosan could be useful for bone trauma management. Copyright © 2014 Elsevier Ltd. All rights reserved.
Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles*S⃞

PubMed Central

Cappuccio, Jenny A.; Blanchette, Craig D.; Sulchek, Todd A.; Arroyo, Erin S.; Kralj, Joel M.; Hinz, Angela K.; Kuhn, Edward A.; Chromy, Brett A.; Segelke, Brent W.; Rothschild, Kenneth J.; Fletcher, Julia E.; Katzen, Federico; Peterson, Todd C.; Kudlicki, Wieslaw A.; Bench, Graham; Hoeprich, Paul D.; Coleman, Matthew A.

2008-01-01

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. PMID:18603642
Prognostic Indications of Elevated MCT4 and CD147 across Cancer Types: A Meta-Analysis

PubMed Central

Bovenzi, Cory D.; Hamilton, James; Tassone, Patrick; Johnson, Jennifer; Cognetti, David M.; Luginbuhl, Adam; Keane, William M.; Zhan, Tingting; Tuluc, Madalina; Bar-Ad, Voichita; Martinez-Outschoorn, Ubaldo; Curry, Joseph M.

2015-01-01

Background. Metabolism in the tumor microenvironment can play a critical role in tumorigenesis and tumor aggression. Metabolic coupling may occur between tumor compartments; this phenomenon can be prognostically significant and may be conserved across tumor types. Monocarboxylate transporters (MCTs) play an integral role in cellular metabolism via lactate transport and have been implicated in metabolic synergy in tumors. The transporters MCT1 and MCT4 are regulated via expression of their chaperone, CD147. Methods. We conducted a meta-analysis of existing publications on the relationship between MCT1, MCT4, and CD147 expression and overall survival and disease-free survival in cancer, using hazard ratios derived via multivariate Cox regression analyses. Results. Increased MCT4 expressions in the tumor microenvironment, cancer cells, or stromal cells were all associated with decreased overall survival and decreased disease-free survival (p < 0.001 for all analyses). Increased CD147 expression in cancer cells was associated with decreased overall survival and disease-free survival (p < 0.0001 for both analyses). Few studies were available on MCT1 expression; MCT1 expression was not clearly associated with overall or disease-free survival. Conclusion. MCT4 and CD147 expression correlate with worse prognosis across many cancer types. These results warrant further investigation of these associations. PMID:26779534
Coatomer subunit beta 2 (COPB2), identified by label-free quantitative proteomics, regulates cell proliferation and apoptosis in human prostate carcinoma cells.

PubMed

Mi, Yuanyuan; Sun, Chuanyu; Wei, Bingbing; Sun, Feiyu; Guo, Yijun; Hu, Qingfeng; Ding, Weihong; Zhu, Lijie; Xia, Guowei

2018-01-01

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G 1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA. Copyright © 2017 Elsevier Inc. All rights reserved.
Overcoming chemotherapy resistance of ovarian cancer cells by liposomal cisplatin: molecular mechanisms unveiled by gene expression profiling.

PubMed

Koch, Martin; Krieger, Michaela L; Stölting, Daniel; Brenner, Norbert; Beier, Manfred; Jaehde, Ulrich; Wiese, Michael; Royer, Hans-Dieter; Bendas, Gerd

2013-04-15

Previously we reported that liposomal cisplatin (CDDP) overcomes CDDP resistance of ovarian A2780cis cancer cells (Krieger et al., Int. J. Pharm. 389, 2010, 10-17). Here we find that the cytotoxic activity of liposomal CDDP is not associated with detectable DNA platination in resistant ovarian cancer cells. This suggests that the mode of action of liposomal CDDP is different from the free drug. To gain insight into mechanisms of liposomal CDDP activity, we performed a transcriptome analysis of untreated A2780cis cells, and A2780cis cells in response to exposure with IC50 values of free or liposomal CDDP. A process network analysis of upregulated genes showed that liposomal CDDP induced a highly different gene expression profile in comparison to the free drug. p53 was identified as a key player directing transcriptional responses to free or liposomal CDDP. The free drug induced expression of essential genes of the intrinsic (mitochondrial) apoptosis pathway (BAX, BID, CASP9) most likely through p38MAPK activation. In contrast, liposomal CDDP induced expression of genes from DNA damage pathways and several genes of the extrinsic pathway of apoptosis (TNFRSF10B-DR5, CD70-TNFSF7). It thus appears that liposomal CDDP overcomes CDDP resistance by inducing DNA damage and in consequence programmed cell death by the extrinsic pathway. Predictions from gene expression data with respect to apoptosis activation were confirmed at the protein level by an apoptosis antibody array. This sheds new light on liposomal drug carrier approaches in cancer and suggests liposomal CDDP as promising strategy for the treatment of CDDP resistant ovarian carcinomas. Copyright © 2013 Elsevier Inc. All rights reserved.
Bacteria Hold Their Breath upon Surface Contact as Shown in a Strain of Escherichia coli, Using Dispersed Surfaces and Flow Cytometry Analysis

PubMed Central

Geng, Jing; Beloin, Christophe; Ghigo, Jean-Marc; Henry, Nelly

2014-01-01

Bacteria are ubiquitously distributed throughout our planet, mainly in the form of adherent communities in which cells exhibit specific traits. The mechanisms underpinning the physiological shift in surface-attached bacteria are complex, multifactorial and still partially unclear. Here we address the question of the existence of early surface sensing through implementation of a functional response to initial surface contact. For this purpose, we developed a new experimental approach enabling simultaneous monitoring of free-floating, aggregated and adherent cells via the use of dispersed surfaces as adhesive substrates and flow cytometry analysis. With this system, we analyzed, in parallel, the constitutively expressed GFP content of the cells and production of a respiration probe—a fluorescent reduced tetrazolium ion. In an Escherichia coli strain constitutively expressing curli, a major E. coli adhesin, we found that single cell surface contact induced a decrease in the cell respiration level compared to free-floating single cells present in the same sample. Moreover, we show here that cell surface contact with an artificial surface and with another cell caused reduction in respiration. We confirm the existence of a bacterial cell “sense of touch” ensuring early signalling of surface contact formation through respiration down modulation. PMID:25054429
Selenite induces posttranscriptional blockade of HLA-E expression and sensitizes tumor cells to CD94/NKG2A-positive NK cells.

PubMed

Enqvist, Monika; Nilsonne, Gustav; Hammarfjord, Oscar; Wallin, Robert P A; Björkström, Niklas K; Björnstedt, Mikael; Hjerpe, Anders; Ljunggren, Hans-Gustaf; Dobra, Katalin; Malmberg, Karl-Johan; Carlsten, Mattias

2011-10-01

CD94/NKG2A is an inhibitory receptor that controls the activity of a large proportion of human NK cells following interactions with the nonclassical HLA class Ib molecule HLA-E expressed on target cells. In this study, we show that selenite (SeO(3)(2-)), an inorganic selenium compound, induces an almost complete loss of cell surface expression of HLA-E on tumor cells of various origins. Selenite abrogated the HLA-E expression at a posttranscriptional level, since selenite exposure led to a dose-dependent decrease in cellular HLA-E protein expression whereas the mRNA levels remained intact. The loss of HLA-E expression following selenite treatment was associated with decreased levels of intracellular free thiols in the tumor cells, suggesting that the reduced HLA-E protein synthesis was caused by oxidative stress. Indeed, HLA-E expression and the level of free thiols remained intact following treatment with selenomethionine, a selenium compound that does not generate oxidative stress. Loss of HLA-E expression, but not of total HLA class I expression, on tumor cells resulted in increased susceptibility to CD94/NK group 2A-positive NK cells. Our results suggest that selenite may be used to potentiate the anti-tumor cytotoxicity in settings of NK cell-based immunotherapies.
A novel packaging system for the generation of helper-free oncolytic MVM vector stocks.

PubMed

Brandenburger, A; Russell, S

1996-10-01

MVM-based autonomous parvoviral vectors have been shown to target the expression of heterologous genes in neoplastic cells and are therefore of interest for cancer gene therapy. The traditional method for production of parvoviral vectors requires the cotransfection of vector and helper plasmids into MVM-permissive cell lines, but recombination between the cotransfected plasmids invariably gives rise to vector stocks that are heavily contaminated with wild-type MVM. Therefore, to minimise recombination between the vector and helper genomes we have utilised a cell line in which the MVM helper functions are expressed inducibly from a modified MVM genome that is stably integrated into the host cell chromosome. Using this MVM packaging cell line, we could reproducibly generate MVM vector stocks that contained no detectable helper virus.
Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

PubMed

Terada, Takaho; Yokoyama, Shigeyuki

2015-01-01

This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.
Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa L.

PubMed

Zirpel, Bastian; Stehle, Felix; Kayser, Oliver

2015-09-01

The Δ9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of Δ9-tetrahydrocannabinolic acid (THCA) using whole cells. Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml(-1) (intracellular) and 44 pkat ml(-1) (extracellular) after 192 h of cultivation at 15 °C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l(-1)) THCA could be produced by 10.5 gCDW l(-1) before enzyme activity was lost. Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production.
Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

PubMed

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

2015-11-10

Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.
Prostaglandin receptor EP3 regulates cell proliferation and migration with impact on survival of endometrial cancer patients

PubMed Central

Zhu, Junyan; Trillsch, Fabian; Mayr, Doris; Kuhn, Christina; Rahmeh, Martina; Hofmann, Simone; Vogel, Marianne; Mahner, Sven; Jeschke, Udo; von Schönfeldt, Viktoria

2018-01-01

Background Prostaglandin E2 (PGE2) receptor 3 (EP3) regulates tumor cell proliferation, migration, and invasion in numerous cancers. The role of EP3 as a prognostic biomarker in endometrial cancer remains unclear. The primary aim of this study was to analyze the prognostic significance of EP3 expression in endometrial cancer. Methods We analyzed the EP3 expression of 140 endometrial carcinoma patients by immunohistochemistry. RL95-2 endometrial cancer cell line was chosen from four endometrial cancer cell lines (RL95-2, Ishikawa, HEC-1-A, and HEC-1-B) according to EP3 expression level. Treated with PGE2 and EP3 antagonist, RL95-2 cells were investigated by MTT, BrdU, and wound healing assay for functional assessment of EP3. Results EP3 staining differed significantly according to WHO tumor grading in both whole cohort (p = 0.01) and the subgroup of endometrioid carcinoma (p = 0.01). Patients with high EP3 expression in their respective tumors had impaired progression-free survival as well as overall survival in both cohorts above. EP3 expression in the overall cohort was identified as an independent prognostic marker for progression-free survival (HR 1.014, 95%CI 1.003-1.024, p = 0.01) when adjusted for age, stage, grading, and recurrence. Treatment with EP3 antagonists induced upregulation of estrogen receptor β and decreased activity of Ras and led to attenuated proliferation and migration of RL95-2 cells. Conclusions EP3 seems to play a crucial role in endometrial cancer progression. In the context of limited systemic treatment options for endometrial cancer, this explorative analysis identifies EP3 as a potential target for diagnostic workup and therapy. PMID:29416671
Prostaglandin receptor EP3 regulates cell proliferation and migration with impact on survival of endometrial cancer patients.

PubMed

Zhu, Junyan; Trillsch, Fabian; Mayr, Doris; Kuhn, Christina; Rahmeh, Martina; Hofmann, Simone; Vogel, Marianne; Mahner, Sven; Jeschke, Udo; von Schönfeldt, Viktoria

2018-01-02

Prostaglandin E2 (PGE2) receptor 3 (EP3) regulates tumor cell proliferation, migration, and invasion in numerous cancers. The role of EP3 as a prognostic biomarker in endometrial cancer remains unclear. The primary aim of this study was to analyze the prognostic significance of EP3 expression in endometrial cancer. We analyzed the EP3 expression of 140 endometrial carcinoma patients by immunohistochemistry. RL95-2 endometrial cancer cell line was chosen from four endometrial cancer cell lines (RL95-2, Ishikawa, HEC-1-A, and HEC-1-B) according to EP3 expression level. Treated with PGE2 and EP3 antagonist, RL95-2 cells were investigated by MTT, BrdU, and wound healing assay for functional assessment of EP3. EP3 staining differed significantly according to WHO tumor grading in both whole cohort (p = 0.01) and the subgroup of endometrioid carcinoma (p = 0.01). Patients with high EP3 expression in their respective tumors had impaired progression-free survival as well as overall survival in both cohorts above. EP3 expression in the overall cohort was identified as an independent prognostic marker for progression-free survival (HR 1.014, 95%CI 1.003-1.024, p = 0.01) when adjusted for age, stage, grading, and recurrence. Treatment with EP3 antagonists induced upregulation of estrogen receptor β and decreased activity of Ras and led to attenuated proliferation and migration of RL95-2 cells. EP3 seems to play a crucial role in endometrial cancer progression. In the context of limited systemic treatment options for endometrial cancer, this explorative analysis identifies EP3 as a potential target for diagnostic workup and therapy.
Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions☆

PubMed Central

Soares, Filipa A.C.; Chandra, Amit; Thomas, Robert J.; Pedersen, Roger A.; Vallier, Ludovic; Williams, David J.

2014-01-01

The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes. PMID:24440272

Differential expression of Mediator complex subunit MED15 in testicular germ cell tumors.

PubMed

Klümper, Niklas; Syring, Isabella; Offermann, Anne; Shaikhibrahim, Zaki; Vogel, Wenzel; Müller, Stefan C; Ellinger, Jörg; Strauß, Arne; Radzun, Heinz Joachim; Ströbel, Philipp; Brägelmann, Johannes; Perner, Sven; Bremmer, Felix

2015-09-17

Testicular germ cell tumors (TGCT) are the most common cancer entities in young men with increasing incidence observed in the last decades. For therapeutic management it is important, that TGCT are divided into several histological subtypes. MED15 is part of the multiprotein Mediator complex which presents an integrative hub for transcriptional regulation and is known to be deregulated in several malignancies, such as prostate cancer and bladder cancer role, whereas the role of the Mediator complex in TGCT has not been investigated so far. Aim of the study was to investigate the implication of MED15 in TGCT development and its stratification into histological subtypes. Immunohistochemical staining (IHC) against Mediator complex subunit MED15 was conducted on a TGCT cohort containing tumor-free testis (n = 35), intratubular germ cell neoplasia unclassified (IGCNU, n = 14), seminomas (SEM, n = 107) and non-seminomatous germ cell tumors (NSGCT, n = 42), further subdivided into embryonic carcinomas (EC, n = 30), yolk sac tumors (YST, n = 5), chorionic carcinomas (CC, n = 5) and teratomas (TER, n = 2). Quantification of MED15 protein expression was performed through IHC followed by semi-quantitative image analysis using the Definiens software. In tumor-free seminiferous tubules, MED15 protein expression was absent or only low expressed in spermatogonia. Interestingly, the precursor lesions IGCNU exhibited heterogeneous but partly very strong MED15 expression. SEM weakly express the Mediator complex subunit MED15, whereas NSGCT and especially EC show significantly enhanced expression compared to tumor-free testis. In conclusion, MED15 is differentially expressed in tumor-free testis and TGCT. While MED15 is absent or low in tumor-free testis and SEM, NSGCT highly express MED15, hinting at the diagnostic potential of this marker to distinguish between SEM and NSGCT. Further, the precursor lesion IGCNU showed increased nuclear MED15 expression in the preinvasive precursor cells, which may provide diagnostic value to distinguish between benign and pre-malignant testicular specimen, and may indicate a role for MED15 in carcinogenesis in TGCT.
Withanolide A Prevents Neurodegeneration by Modulating Hippocampal Glutathione Biosynthesis during Hypoxia

PubMed Central

Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

2014-01-01

Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner. PMID:25310001
Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia.

PubMed

Baitharu, Iswar; Jain, Vishal; Deep, Satya Narayan; Shroff, Sabita; Sahu, Jayanta Kumar; Naik, Pradeep Kumar; Ilavazhagan, Govindasamy

2014-01-01

Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner.
Insect cells as factories for biomanufacturing.

PubMed

Drugmand, Jean-Christophe; Schneider, Yves-Jacques; Agathos, Spiros N

2012-01-01

Insect cells (IC) and particularly lepidopteran cells are an attractive alternative to mammalian cells for biomanufacturing. Insect cell culture, coupled with the lytic expression capacity of baculovirus expression vector systems (BEVS), constitutes a powerful platform, IC-BEVS, for the abundant and versatile formation of heterologous gene products, including proteins, vaccines and vectors for gene therapy. Such products can be manufactured on a large scale thanks to the development of efficient and scaleable production processes involving the integration of a cell growth stage and a stage of cell infection with the recombinant baculovirus vector. Insect cells can produce multimeric proteins functionally equivalent to the natural ones and engineered vectors can be used for efficient expression. Insect cells can be cultivated easily in serum- and protein-free media. A growing number of companies are currently developing an interest in producing therapeutics using IC-BEVS, and many products are today in clinical trials and on the market for veterinary and human applications. This review summarizes current knowledge on insect cell metabolism, culture conditions and applications. Copyright © 2011 Elsevier Inc. All rights reserved.
A Novel Molecular Targeting of a Tumor-Specific Oncogenic Mutant Receptor in Human Prostate Cancer

DTIC Science & Technology

2005-02-01

in cells and can generate dominant negative mutant (15). Hammerhead ribozymes are self-cleaving RNAs whose catalytic activity has been mapped to a...specific ribozyme targeted at the fusion junction of EGFRvIII. This specific EGFRvIII ribozyme is able to effectively cleave EGFRvIII mRNA under...physiological conditions in a cell-free system. While expressing this EGFRvIII- ribozyme in 32D/EGFRvIII cell, EGFRvIII- ribozyme is capable of down-regulating
Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS− cell populations

PubMed Central

El-Sayed, Karim M Fawzy; Paris, Sebastian; Graetz, Christian; Kassem, Neemat; Mekhemar, Mohamed; Ungefroren, Hendrick; Fändrich, Fred; Dörfer, Christof

2015-01-01

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells. PMID:25257881
Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs.

PubMed

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

2017-01-01

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

PubMed

Lobo, Nazleen C; Gedye, Craig; Apostoli, Anthony J; Brown, Kevin R; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A S; Ailles, Laurie

2016-07-16

Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability to establish primary cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients.
Stearoyl-CoA desaturase activity in bovine cumulus cells protects the oocyte against saturated fatty acid stress

PubMed Central

Aardema, Hilde; van Tol, Helena T. A.; Wubbolts, Richard W.; Brouwers, Jos F. H. M.; Gadella, Bart M.; Roelen, Bernard A. J.

2017-01-01

Abstract Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid. PMID:28486699
N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

PubMed

Ojima-Kato, Teruyo; Nagai, Satomi; Nakano, Hideo

2017-05-01

Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
High expression of CXCR4, CXCR7 and SDF-1 predicts poor survival in renal cell carcinoma

PubMed Central

2012-01-01

Background Chemokines and their receptors are known to play important roles in the tumorigenesis of many malignancies. The aim of this study was to evaluate the prognostic impact of the expression of the chemokine SDF-1 and its receptors CXCR4 and CXCR7 in patients with renal cell carcinoma. Methods The expression of CXCR4, CXCR7 and SDF-1 in specimens from 97 renal cell carcinoma patients was evaluated by immunohistochemistry on a tissue microarray. These results were correlated with the clinicopathological parameters and survival of the patients. Results CXCR4 and CXCR7 were expressed in all patients, whereas SDF-1 was expressed in 61 patients (62.9%). No association was observed between the expression of CXCR4, CXCR7 or SDF-1 and the clinical or pathological data except between SDF-1 expression and Fuhrman’s grade (P = 0.015). Patients with high expression of CXCR4, CXCR7 and SDF-1 had shorter overall survival and recurrence-free survival than those with low expression. In a multivariate analysis, the high expression of CXCR4, CXCR7 and SDF-1 correlated with poor overall survival and recurrence-free survival independent of gender, age, AJCC stage, lymph node status, metastasis, histologic variant and Fuhrman’s grade. Conclusions High levels of CXCR4, CXCR7 and SDF-1 were associated with poor overall survival and recurrence-free survival in renal cell carcinoma patients. CXCR4, CXCR7 and SDF-1 may serve as useful prognostic markers and therapeutic targets for renal cell carcinoma. PMID:23039915
Effect of silibinin-loaded nano-niosomal coated with trimethyl chitosan on miRNAs expression in 2D and 3D models of T47D breast cancer cell line.

PubMed

Yazdi Rouholamini, Seyede Elmira; Moghassemi, Saeid; Maharat, Zahra; Hakamivala, Amirhossien; Kashanian, Susan; Omidfar, Kobra

2018-05-01

Silibinin is a natural flavonoid with a strong antioxidant property and weak cytotoxic activity. It has demonstrated anti-tumoural activity against many types of malignancies; however, due to its hydrophobic structure, it has poor water solubility, bioavailability and permeability across intestinal epithelial cells. To improve the effect of silibinin, we have vehiculated silibinin by a highly stable niosomal nanostructure based on a Span 60/cholesterol (CH)/N-trimethyl chitosan (TMC) system in order to study its potential application for the delivery of silibinin in T47D cultured under three-dimensional (3D) and two-dimensional (2D) conditions. To study the effect of nanodrug on miRNAs expression, we evaluated quantitative expression of miRNA-21 and miRNA-15a as well as miR-141 and miR-200c which act as oncogene and tumour suppressors by real-time PCR. Results demonstrated that the mechanism of nanodrug action as well as the response of tumour cells differed in 3D culture as compared to 2D. Delivery of silibinin-loaded niosomes coated with TMC was found to be more effective in inhibiting the growth of tumour cells and inducing apoptosis than free silibinin administration. In silibinin-treated cells, death occurred in a dose- and time- dependent manner by induction of apoptosis and alteration of the cell cycle. Real-time PCR analysis revealed a decrease in miR-21, miR-15a and miR-141while increase in miR-200c expression levels was observed in silibinin-treated cells relative to the levels in the untreated cells. The results show that nanodrug delivery was more effective than free silibinin administration in changing the level of miRNAs expression in cancer cells. Therefore, niosomal nanostructure with TMC could be a suitable vehicle for hydrophobic compounds, such as silibinin, by improving their action in cancer therapy.
Prognostic Impact of Activated Leucocyte Cell Adhesion Molecule (ALCAM/CD166) in Infantile Neuroblastoma.

PubMed

Wachowiak, Robin; Mayer, Steffi; Kaifi, Jussuf; Gebauer, Florian; Izbicki, Jakob R; Lacher, Martin; Bockhorn, Maximilian; Tachezy, Michael

2016-08-01

Activated leukocyte cell adhesion molecule (ALCAM/CD166) as a member of the 'immunoglobulin superfamily' is known to be involved in cancer cell proliferation and migration. The aim of this study was to investigate the expression of ALCAM in neuroblastoma tissues. ALCAM expression was analyzed in primary neuroblastoma specimens by immunohistochemistry on microarray sections. Histopathological and clinical data were correlated with ALCAM expression and survival analysis was performed. Sixty-six children were included in the study. Strong expression of ALCAM was detected in 52 (79%) of the samples. Weak expression was significantly correlated with the International Neuroblastoma Staging System (INSS) stage (p=0.024) and positive n-MYC amplification (p=0.019). Recurrence-free survival (RFS) and overall survival (OS) were significantly shorter if ALCAM was expressed weakly (p=0.032 and p=0.001). Weak ALCAM expression was significantly correlated with established markers for poor prognosis, as well as shorter RFS and OS. ALCAM might be considered as a prognostic marker for infantile neuroblastoma. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells.

PubMed

Brown, Rachel C; Morris, Andrew P; O'Neil, Roger G

2007-01-26

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.
TIGHT JUNCTION PROTEIN EXPRESSION AND BARRIER PROPERTIES OF IMMORTALIZED MOUSE BRAIN MICROVESSEL ENDOTHELIAL CELLS

PubMed Central

Brown, Rachel C.; Morris, Andrew P.; O’Neil, Roger G.

2007-01-01

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions. PMID:17169347
Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

PubMed

Qu, Su; Yan, Liang; Fang, Bo; Ye, Shoudong; Li, Ping; Ge, Shengyang; Wu, Jian; Qu, Di; Song, Houyan

2017-04-15

To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system. Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test. Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition. This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation. Copyright © 2017 Elsevier Inc. All rights reserved.
A New Cell-Free System to Study BRCA1 Function

DTIC Science & Technology

2014-05-01

antibodies to FANCI, FANCD2, DNA pol e, FANCA , FANCM. We first wanted to test whether Approach, which is inhibited in BRCA1-depleted egg extracts...analysis. We have not initiated this task. 4c. For novel proteins whose binding to chromatin depends on BRCA1: clone the gene , express the protein
NDRG2 correlated with favorable recurrence-free survival inhibits metastasis of mouse breast cancer cells via attenuation of active TGF-β production.

PubMed

Oh, Sang-seok; Kim, Donghyeok; Kim, Dong-Hee; Chang, Hong Hee; Sohn, Kyung-Cheol; Kim, Kyo Hyun; Jung, Sung Hoo; Lee, Byoung Kil; Kim, Joo Heon; Kim, Kwang Dong

2012-10-01

N-myc downstream-regulated gene 2 (NDRG2) has been studied for its inhibitory effects against growth and metastasis of many tumor cell types. In this study, we showed NDRG2 expression was correlated with favorable recurrence-free survival of patients with breast cancer and inhibited metastasis of breast cancer cells (4T1). NDRG2 expression was examined in 189 breast carcinoma tissues and paired normal breast tissues using immunohistochemistry. Histological and clinicopathological data were correlated using Pearson's chi-square test of independence. NDRG2 expression in human breast cancer tissues was inversely associated with lymph node metastasis and pTNM stage. Furthermore, patients with breast cancer with a high level of NDRG2 expression showed favorable recurrence-free survival (P = 0.038). To study the effect of NDRG2 on metastasis in vivo, we established an NDRG2-overexpressing mouse breast cancer cell line (4T1-NDRG2) and measured the metastasis and survival of 4T1-NDRG2 tumor-bearing mice. To test whether transforming growth factor β (TGF-β)- mediated metastasis of 4T1 was inhibited by NDRG2 expression, TGF-Smad-binding element (SBE)-luciferase activity and/or measurement of active TGF-β were performed in cell or tumor tissue level. 4T1-NDRG2 cells grew gradually and showed less metastatic activity in vivo and low invasiveness in vitro. 4T1-NDRG2 cells showed lower SBE-luciferase activity and lower level of active autocrine TGF-β than 4T1-Mock did. Correctly, our data show that NDRG2 significantly suppress tumor metastasis by attenuating active autocrine TGF-β production, and the attenuation might be typically associated with the favorable recurrence-free survival of patients clinically.
Animal serum-free expansion and differentiation of human mesenchymal stromal cells.

PubMed

Felka, Tino; Schäfer, Richard; De Zwart, Peter; Aicher, Wilhelm K

2010-04-01

Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.
The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH.

PubMed

Barbero-Becerra, Varenka J; Giraudi, Pablo J; Chávez-Tapia, Norberto C; Uribe, Misael; Tiribelli, Claudio; Rosso, Natalia

2015-10-01

A complex interplay exists between hepatocytes and hepatic stellate cells (HSC) in hepatic fibrogenesis. The activation of HSCs after liver injury leads to production of extracellular matrix (ECM). Co-culture models could be useful to mimic the liver microenvironment. This study evaluates the effect of free fatty acids (FFA) on HSC cells and the interplay with hepatocytes via both soluble-mediator and cell-cell contact. The human hepatocyte cell line (HuH7) and HSC cells (LX2) were exposed to FFA for 24 h in 3 different experimental set-ups: (A) monoculture of HSC; (B) Transwell® system (effect of soluble mediators); and (C) Simultaneous Co-Culture (SCC) (cell-to-cell connections). Intracellular FFA accumulation was assessed qualitatively (microscopy) and quantitatively (flow cytometry); the activation of HSC (alpha smooth muscle actin, α-SMA) expression of ECM components were quantified by RT-PCR. FFA exposure induces intracellular fat accumulation in all the experimental set-up but the expression of α-SMA was significantly increased only in SCC. On the contrary, the expression of ECM was substantially decreased in the transwell® system. The HSC activation is independent of FFA accumulation but requires cell-to-cell interaction with hepatocyte. On the contrary, the gene regulation of ECM components seems to occur through paracrine mediators. Copyright © 2015 Elsevier Ltd. All rights reserved.

Part I. Development of a model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell carcinoma cell lines.

PubMed

Yarmolyuk, Yaroslav R; Vesper, Benjamin J; Paradise, William A; Elseth, Kim M; Tarjan, Gabor; Haines, G Kenneth; Radosevich, James A

2011-02-01

The free radical nitric oxide (NO) is over-expressed in many tumors, including head and neck squamous cell carcinomas (HNSCC); however, the role NO plays in tumor pathophysiology is still not well understood. We, herein, report the development of an in vitro model system which can be used to probe the role of NO in the carcinogenesis of HNSCC. Five HNSCC cell lines were adapted to a high NO (HNO) environment by gradually introducing increasing concentrations of DETA-NONOate, a nitrogen-based NO donor, to cell media. The adaptation process was carried out until a sufficiently high enough donor concentration was reached which enabled the HNO cells to survive and grow, but which was lethal to the original, unadapted ("parent") cells. The adapted HNO cells exhibited analogous morphology to the parent cells, but grew better than their corresponding parent cells in normal media, on soft agar, and in the presence of hydrogen peroxide, an oxygen-based free radical donor. These results indicate that the HNO cell lines are unique and possess biologically different properties than the parent cell lines from which they originated. The HNO/parent cell lines developed herein may be used as a model system to better understand the role NO plays in HNSCC carcinogenesis.
Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

NASA Astrophysics Data System (ADS)

Lin, Wen Jen; Chien, Wei Hsuan

2015-09-01

The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly( d,l-lactide- co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.
Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

PubMed

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.
Redox sensor CtBP mediates hypoxia-induced tumor cell migration

PubMed Central

Zhang, Qinghong; Wang, Su-Yan; Nottke, Amanda C.; Rocheleau, Jonathan V.; Piston, David W.; Goodman, Richard H.

2006-01-01

The rapid growth and poor vascularization of solid tumors expose cancer cells to hypoxia, which promotes the metastatic phenotype by reducing intercellular adhesion and increasing cell motility and invasiveness. In this study, we found that hypoxia increased free NADH levels in cancer cells, promoting CtBP recruitment to the E-cadherin promoter. This effect was blocked by pyruvate, which prevents the NADH increase. Furthermore, hypoxia repressed E-cadherin gene expression and increased tumor cell migration, effects that were blocked by CtBP knockdown. We propose that CtBP senses levels of free NADH to control expression of cell adhesion genes, thereby promoting tumor cell migration under hypoxic stress. PMID:16740659
Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks.

PubMed

Poojan, Shiv; Kim, Han-Seong; Yoon, Ji-Woon; Sim, Hye Won; Hong, Kyeong-Man

2018-05-20

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.
Cell-free extracts of Propionibacterium acnes stimulate cytokine production through activation of p38 MAPK and Toll-like receptor in SZ95 sebocytes.

PubMed

Huang, Yu-Chun; Yang, Chao-Hsun; Li, Ting-Ting; Zouboulis, Christos C; Hsu, Han-Chi

2015-10-15

Propionibacterium acnes has been considered to influence the acne lesions. The present study intended to elucidate the underlying signaling pathways of P. acnes in human sebaceous gland cells relative to the generation of proinflammatory cytokines. Cell-free extracts of P. acnes under stationary growth phase were co-incubated with human immortalized SZ95 sebocytes. Then, cell-free P. acnes extracts-induced cytokine expression was evaluated by measuring mRNA and protein levels using quantitative RT-PCR and ELISA. Changes of phosphorylated cell signaling proteins and transcription factors were measured by Western blots and Milliplex assay. The interactive molecular mechanisms of P. acnes and sebocytes were examined through use of shRNA and the specific inhibitors of signaling pathways. Cell-free extracts of P. acnes significantly stimulated secretion of interleukin (IL)-8 and IL-6 in SZ95 sebocytes. The degradation of IκB-α and increased phosphorylation of IκB-α, p38 mitogen activated protein kinase (MAPK), CREB, and STAT3 were demonstrated. Quantitative RT-PCR measurements revealed that gene expression of IL-8 and Toll-like receptor 2 (TLR2) was enhanced by cell-free extracts of P. acnes. In addition, the NF-κB inhibitor BMS345541, p38 MAPK inhibitor SB203580, or anti-TLR2 neutralizing antibody prevented cell-free P. acnes extracts-induced secretion of IL-8. Knockdown of TLR2 using shRNA exerted similar inhibitory effects on IL-8 expression. Moreover, inhibition of STAT3 activity by STA-21 enhanced P. acnes-mediated secretion of IL-8. Cell-free extracts of P. acnes are capable to activate NF-κB and p38 MAPK pathways and up-regulate secretion of IL-8 through TLR2-dependent signaling in human SZ95 sebocytes. Copyright © 2015 Elsevier Inc. All rights reserved.
Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease

PubMed Central

Jison, Maria L.; Munson, Peter J.; Barb, Jennifer J.; Suffredini, Anthony F.; Talwar, Shefali; Logun, Carolea; Raghavachari, Nalini; Beigel, John H.; Shelhamer, James H.; Danner, Robert L.; Gladwin, Mark T.

2016-01-01

In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury. PMID:15031206
Kinetic Self-Assembly of DNA Tiles and Bricks

DTIC Science & Technology

2016-08-26

research, and educa- tion. This is achieved by freeze drying cell-free systems into paper and other porous substrates to create materials with the...different toehold switches on paper . Experiments were performed by freeze drying the recombinant PT7 expression system onto paper discs, along with linear...DNA encoding specific switch RNAs. The paper discs were rehy- drated with or without the complementary RNA trigger 24 hr after drying and then
Cell-free formation and interactome analysis of caveolae.

PubMed

Jung, WooRam; Sierecki, Emma; Bastiani, Michele; O'Carroll, Ailis; Alexandrov, Kirill; Rae, James; Johnston, Wayne; Hunter, Dominic J B; Ferguson, Charles; Gambin, Yann; Ariotti, Nicholas; Parton, Robert G

2018-06-04

Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae ( Lt ) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1 Y14 )-stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1. © 2018 Jung et al.
Recombinant interferon-gamma secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium.

PubMed

Mols, J; Peeters-Joris, C; Wattiez, R; Agathos, S N; Schneider, Y-J

2005-01-01

Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.
Serum- and Growth-Factor-Free Three-Dimensional Culture System Supports Cartilage Tissue Formation by Promoting Collagen Synthesis via Sox9–Col2a1 Interaction

PubMed Central

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

2014-01-01

Objective: One of the factors preventing clinical application of regenerative medicine to degenerative cartilage diseases is a suitable source of cells. Chondrocytes, the only cell type of cartilage, grown in vitro under culture conditions to expand cell numbers lose their phenotype along with the ability to generate hyaline cartilaginous tissue. In this study we determine that a serum- and growth-factor-free three-dimensional (3D) culture system restores the ability of the passaged chondrocytes to form cartilage tissue in vitro, a process that involves sox9. Methods: Bovine articular chondrocytes were passaged twice to allow for cell number expansion (P2) and cultured at high density on 3D collagen-type-II-coated membranes in high glucose content media supplemented with insulin and dexamethasone (SF3D). The cells were characterized after monolayer expansion and following 3D culture by flow cytometry, gene expression, and histology. The early changes in signaling transduction pathways during redifferentiation were characterized. Results: The P2 cells showed a progenitor-like antigen profile of 99% CD44+ and 40% CD105+ and a gene expression profile suggestive of interzone cells. P2 in SF3D expressed chondrogenic genes and accumulated extracellular matrix. Downregulating insulin receptor (IR) with HNMPA-(AM3) or the PI-3/AKT kinase pathway (activated by insulin treatment) with Wortmannin inhibited collagen synthesis. HNMPA-(AM3) reduced expression of Col2, Col11, and IR genes as well as Sox6 and -9. Co-immunoprecipitation and chromatin immunoprecipitation analyses of HNMPA-(AM3)-treated cells showed binding of the coactivators Sox6 and Med12 with Sox9 but reduced Sox9–Col2a1 binding. Conclusions: We describe a novel culture method that allows for increase in the number of chondrocytes and promotes hyaline-like cartilage tissue formation in part by insulin-mediated Sox9–Col2a1 binding. The suitability of the tissue generated via this approach for use in joint repair needs to be examined in vivo. PMID:24606204
Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme.

PubMed Central

Holm, P. S.; Scanlon, K. J.; Dietel, M.

1994-01-01

A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line. Images Figure 1 Figure 3 Figure 2 PMID:7914421
Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

PubMed

Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

2017-07-25

Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of the inducible vector using the same promoter. Finally, we used gfp as a reporter gene in combination with the two promoters Pgrac01 and Pgrac100 to test the new vector types. The GFP expression levels could be repressed at least 1.5 times for the Pgrac01-gfp+ inducer-free construct in E. coli. The inducer-free constructs Pgrac01-gfp+ and Pgrac100-gfp+ allowed GFP expression at high levels from 23 × 10 4 to 32 × 10 4 RFU units and 9-13% of total intracellular proteins. We could reconfirm the two major advantages of the new inducer-free expression plasmids: (1) Strong repression of the target gene expression in the E. coli cloning strain, and (2) production of the target protein at high levels in B. subtilis in the absence of the inducer. We propose a general strategy to generate inducer-free expression vector by using IPTG-inducible vectors, and more specifically we developed inducer-free expression plasmids using IPTG-inducible promoters in the absence of the LacI repressor. These plasmids could be an excellent choice for high-level production of recombinant proteins in B. subtilis without the addition of inducer and at the same time maintaining a low basal level of the recombinant proteins in E. coli. The repression of the recombinant gene expression would facilitate cloning of genes that potentially inhibit the growth of E. coli cloning strains. The inducer-free expression plasmids will be extended versions of the current available IPTG-inducible expression vectors for B. subtilis, in which all these vectors use the same cognate promoters. These inducer-free and previously developed IPTG-inducible expression plasmids will be a useful cassette to study gene expression at a small scale up to a larger scale up for the production of recombinant proteins.
Cancer stem-like cells of ovarian clear cell carcinoma are enriched in the ALDH-high population associated with an accelerated scavenging system in reactive oxygen species.

PubMed

Mizuno, T; Suzuki, N; Makino, H; Furui, T; Morii, E; Aoki, H; Kunisada, T; Yano, M; Kuji, S; Hirashima, Y; Arakawa, A; Nishio, S; Ushijima, K; Ito, K; Itani, Y; Morishige, K

2015-05-01

In ovarian cancer cases, recurrence after chemotherapy is frequently observed, suggesting the involvement of ovarian cancer stem-like cells (CSCs). The chemoresistance of ovarian clear cell carcinomas is particularly strong in comparison to other epithelial ovarian cancer subtypes. We investigated the relationship between a CSC marker, aldehyde dehydrogenase 1 (ALDH1), and clinical prognosis using ovarian clear cell carcinoma tissue samples. Furthermore, we investigated the antioxidant mechanism by which CSCs maintain a lower reactive oxygen species (ROS) level, which provides protection from chemotherapeutic agents. Immunohistochemical staining was performed to examine the CSC markers (CD133, CD44, ALDH1) using ovarian clear cell carcinoma tissue samples (n=81). Clear cell carcinoma cell lines (KOC-7C, OVTOKO) are separated into the ALDH-high and ALDH-low populations by ALDEFLUOR assay and fluorescence-activated cell sorting (FACS). We compared the intracellular ROS level, mRNA level of the antioxidant enzymes and Nrf2 expression of the two populations. High ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. ALDH1 expression significantly reduced progression free survival. Other markers are not related to clinical stage and prognosis. ALDH-high cells contained a lower ROS level than ALDH-low cells. Antioxidant enzymes were upregulated in ALDH-high cells. ALDH-high cells showed increased expression of Nrf2, a key transcriptional factor of the antioxidant system. ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemoresistance in ovarian clear cell carcinoma. Copyright © 2014 Elsevier Inc. All rights reserved.
Increased expression of human leucocyte antigen class I free heavy chains on monocytes of patients with spondyloarthritis and cells transfected with HLA-B27.

PubMed

Ding, Jin; Feng, Yuan; Zheng, Zhao Hui; Li, Xue Yi; Wu, Zhen Biao; Zhu, Ping

2015-02-01

Human leucocyte antigen (HLA)-B27 expression is correlated with spondyloarthritis (SpA), but its role in disease pathogenesis remains unclear. The aim of the study was to determine whether HLA-B27 free heavy chain (FHC) contributes to SpA pathogenesis. Flow cytometry was used to analyse the FHC expression on CD3+ and CD14+ cells in the peripheral blood (PB) and synovial fluid (SF) from SpA patients, healthy controls, and rheumatoid arthritis (RA) patients. Human monocytic U937 cell lines stably expressing enhanced green fluorescence protein (EGFP)/HLA-B27, EGFP/HLA-A2 or EGFP alone were created to further investigate the relation between HLA-B27 and FHC expression. The relative FHC level on CD14+ PB cells was significantly higher in SpA patients than in controls, but lower than on the SF cells of SpA patients. No significant correlation was found for relative FHC expression with HLA-B27 or β2-microglobulin expression. HLA-B27-transfected U937 cells expressed higher FHC levels than either EGFP/HLA-A2- or EGFP-transfected cells. HLA class I FHC expression was significantly increased on monocytes of SpA patients and HLA-B27-transfected cells, implying that FHC, perhaps mostly derived from HLA-B27, plays an important role in SpA pathogenesis. © 2014 John Wiley & Sons Ltd.
The prognostic role of CD68 and FoxP3 expression in patients with primary central nervous system lymphoma.

PubMed

Cho, Hyunsoo; Kim, Se Hoon; Kim, Soo-Jeong; Chang, Jong Hee; Yang, Woo Ick; Suh, Chang-Ok; Cheong, June-Won; Kim, Yu Ri; Lee, Jung Yeon; Jang, Ji Eun; Kim, Yundeok; Min, Yoo Hong; Kim, Jin Seok

2017-07-01

The prognostic role of CD68 and FoxP3 in primary central nervous system lymphoma (PCNSL) has not been evaluated. Thus, we examined the prognostic significance of CD68 and FoxP3 expression in tumor samples of 76 newly diagnosed immunocompetent PCNSL patients. All patients were treated initially with high-dose methotrexate (HD-MTX)-based chemotherapy, and 16 (21.1%) patients received upfront autologous stem cell transplantation (ASCT) consolidation. High expression of CD68 (>55 cells/high-power field) or FoxP3 (>15 cells/high-power field) was observed in 10 patients, respectively. High CD68 expression was associated with inferior overall survival (OS) and progression-free survival (PFS) in multivariate analysis (P = 0.023 and P = 0.021, respectively). In addition, we performed subgroup analysis based on upfront ASCT. High CD68 expression was also associated with inferior OS and PFS in multivariate analysis (P = 0.013 and P < 0.001, respectively) among patients who did not receive upfront ASCT (n = 60), but not in patients who received upfront ASCT. The expression of FoxP3 was not significantly associated with survival. Therefore, we identified a prognostic significance of high CD68 expression in PCNSL, which suggests a need for further clinical trials and biological studies on the role of PCNSL tumor microenvironment.
Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

PubMed

Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

2016-08-01

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices.

PubMed

Josephs, S F; Loudovaris, T; Dixit, A; Young, S K; Johnson, R C

1999-01-01

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.
Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

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

Coleman, Matthew A.; Cappuccio, Jenny A.; Blanchette, Craig D.

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteinsmore » as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.« less
Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

DOE PAGES

Coleman, Matthew A.; Cappuccio, Jenny A.; Blanchette, Craig D.; ...

2016-03-25

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteinsmore » as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.« less

Glutathione S-transferase Pi expression in invasive breast cancer and its relation with the clinical outcome.

PubMed

Franco, R L; Schenka, N G M; Schenka, A A A; Rezende, L F; Gurgel, M S C

2012-01-01

Glutathione S-transferase (GST) is a cytosolic enzymatic system involved in cellular detoxifying process. In vitro studies have shown that the presence of this enzymatic system in breast carcinoma cells can accelerate the elimination of drugs commonly used in chemotherapy, thereby decreasing its efficacy. The aim of the present study was to evaluate the association between GST Pi expression by breast carcinoma cells and disease-free and overall survival. Ninety-five female patients with invasive breast carcinoma submitted to surgical treatment and adjuvant chemotherapy from January, 1995 to June, 1997 and followed until August, 2006 were evaluated. The expression of GST Pi in breast carcinoma cells, determined by immunohistochemistry, was correlated with several clinical and pathological parameters of prognostic significance. There were 36 (37.9%) GST Pi-positive cases. GST Pi immunoexpression was not significantly correlated with patient's age, histological tumor type, clinical stage, hormone receptor status and survival. On the other hand, GST Pi positivity showed a significant correlation with a lower histological grade/C-erb-B2 negative breast carcinoma phenotype. The findings suggest that GST Pi expression does not constitute a satisfactory prognostic factor in breast cancer.
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.
Lymphoblast-derived integration-free iPSC line AD-TREM2-3 from a 74 year-old Alzheimer's disease patient expressing the TREM2 p.R47H variant.

PubMed

Martins, Soraia; Yigit, Hatice; Bohndorf, Martina; Graffmann, Nina; Fiszl, Aurelian Robert; Wruck, Wasco; Sleegers, Kristel; Van Broeckhoven, Christine; Adjaye, James

2018-06-01

Human lymphoblast cells from a male diagnosed with Alzheimer's disease (AD) expressing the TREM2 p.R47H variant were used to generate integration-free induced pluripotent stem cells (iPSCs) by over-expressing episomal-based plasmids harbouring OCT4, SOX2, KLF4, LIN28, L-MYC and p53 shRNA. The derived iPSC line - AD-TREM2-3 was defined as pluripotent based on (i) expression of pluripotency-associated markers (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptome of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.940. Copyright © 2018. Published by Elsevier B.V.
Wash-free and selective imaging of epithelial cell adhesion molecule (EpCAM) expressing cells with fluorogenic peptide ligands.

PubMed

K C, Tara Bahadur; Suga, Kanako; Isoshima, Takashi; Aigaki, Toshiro; Ito, Yoshihiro; Shiba, Kiyotaka; Uzawa, Takanori

2018-06-02

Detection of the cells expressing an epithelial cell adhesion molecule (EpCAM) is a crucial step to identify circulating tumor cells (CTCs) from blood. To detect the EpCAM, we here designed and synthesized a series of fluorogenic peptides. Specifically, we functionalized an EpCAM-binding peptide, Ep114, by replacing its amino acids to an aminophenylalanine that was modified with environmentally sensitive 7-nitro-2,1,3-benzoxadiazole (NBD-amPhe). Among six synthesized peptides, we have found that two peptides, Q4X and V6X (X represents NBD-amPhe), retain the Ep114's binding ability and specifically mark EpCAM-expressing cells by just adding these peptides to the cultivation medium. Our wash-free, fluorogenic peptide ligands would boost the development of next generation devices for CTC diagnoses. Copyright © 2018 Elsevier Inc. All rights reserved.
Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

PubMed

Schmidt, Ronny; Cook, Elizabeth A; Kastelic, Damjana; Taussig, Michael J; Stoevesandt, Oda

2013-08-02

We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes. Copyright © 2013 Elsevier B.V. All rights reserved.
Genome-wide screen identifies a novel prognostic signature for breast cancer survival

DOE PAGES

Mao, Xuan Y.; Lee, Matthew J.; Zhu, Jeffrey; ...

2017-01-21

Large genomic datasets in combination with clinical data can be used as an unbiased tool to identify genes important in patient survival and discover potential therapeutic targets. We used a genome-wide screen to identify 587 genes significantly and robustly deregulated across four independent breast cancer (BC) datasets compared to normal breast tissue. Gene expression of 381 genes was significantly associated with relapse-free survival (RFS) in BC patients. We used a gene co-expression network approach to visualize the genetic architecture in normal breast and BCs. In normal breast tissue, co-expression cliques were identified enriched for cell cycle, gene transcription, cell adhesion,more » cytoskeletal organization and metabolism. In contrast, in BC, only two major co-expression cliques were identified enriched for cell cycle-related processes or blood vessel development, cell adhesion and mammary gland development processes. Interestingly, gene expression levels of 7 genes were found to be negatively correlated with many cell cycle related genes, highlighting these genes as potential tumor suppressors and novel therapeutic targets. A forward-conditional Cox regression analysis was used to identify a 12-gene signature associated with RFS. A prognostic scoring system was created based on the 12-gene signature. This scoring system robustly predicted BC patient RFS in 60 sampling test sets and was further validated in TCGA and METABRIC BC data. Our integrated study identified a 12-gene prognostic signature that could guide adjuvant therapy for BC patients and includes novel potential molecular targets for therapy.« less
Genome-wide screen identifies a novel prognostic signature for breast cancer survival

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

Mao, Xuan Y.; Lee, Matthew J.; Zhu, Jeffrey

Large genomic datasets in combination with clinical data can be used as an unbiased tool to identify genes important in patient survival and discover potential therapeutic targets. We used a genome-wide screen to identify 587 genes significantly and robustly deregulated across four independent breast cancer (BC) datasets compared to normal breast tissue. Gene expression of 381 genes was significantly associated with relapse-free survival (RFS) in BC patients. We used a gene co-expression network approach to visualize the genetic architecture in normal breast and BCs. In normal breast tissue, co-expression cliques were identified enriched for cell cycle, gene transcription, cell adhesion,more » cytoskeletal organization and metabolism. In contrast, in BC, only two major co-expression cliques were identified enriched for cell cycle-related processes or blood vessel development, cell adhesion and mammary gland development processes. Interestingly, gene expression levels of 7 genes were found to be negatively correlated with many cell cycle related genes, highlighting these genes as potential tumor suppressors and novel therapeutic targets. A forward-conditional Cox regression analysis was used to identify a 12-gene signature associated with RFS. A prognostic scoring system was created based on the 12-gene signature. This scoring system robustly predicted BC patient RFS in 60 sampling test sets and was further validated in TCGA and METABRIC BC data. Our integrated study identified a 12-gene prognostic signature that could guide adjuvant therapy for BC patients and includes novel potential molecular targets for therapy.« less
Compound Danshen injection improves endotoxin-induced microcirculatory disturbance in rat mesentery

PubMed Central

Han, Jing-Yan; Horie, Yoshinori; Miura, Soichiro; Akiba, Yasutada; Guo, Jun; Li, Dan; Fan, Jing-Yu; Liu, Yu-Ying; Hu, Bai-He; An, Li-Hua; Chang, Xin; Xu, Man; Guo, De-An; Sun, Kai; Yang, Ji-Ying; Fang, Shu-Ping; Xian, Ming-Ji; Kizaki, Masahiro; Nagata, Hiroshi; Hibi, Toshifumi

2007-01-01

AIM: To investigate the effect of compound Danshen injection on lipopolysaccharide (LPS)-induced rat mesenteric microcirculatory dysfunctions and the underlying possible mechanism by an inverted intravital microscope and high-speed video camera system. METHODS: LPS was continuously infused through the jugular artery of male Wistar rats at the dose of 2 mg/kg per hour. Changes in mesenteric microcirculation, such as diameters of arterioles and venules, velocity of RBCs in venules, leukocyte rolling, adhesion and emigration, free radicals released from post-capillary venules, FITC-albumin leakage and mast cell degranulation, were observed through an inverted intravital microscope assisted with CCD camera and SIT camera. Meanwhile, the expression of adhesion molecules CD11b/CD18 and the production of free radical in neutrophils, and the expression of intercellular adhesion molecule 1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs) were quantified by flow cytometry (FACS) in vitro. RESULTS: The continuous infusion with LPS resulted in a number of responses in microcirculation, including a significant increase in the positive region of venule stained with Monastral blue B, rolling and adhesion of leukocytes, production of oxygen radical in venular wall, albumin efflux and enhanced mast cell degranulation in vivo, all of which, except for the leukocyte rolling, were attenuated by the treatment with compound Danshen injection. Experiments performed in vitro further revealed that the expression of CD11b/CD18 and the production of oxygen free radical in neutrophils, and the expression of ICAM-1 in HUVECs were increased by exposure to LPS, and they were attenuated by compound Danshen injection. CONCLUSION: These results suggest that compound Danshen injection is an efficient drug with multi-targeting potential for improving the microcirculatory disturbance. PMID:17659708
Compound Danshen injection improves endotoxin-induced microcirculatory disturbance in rat mesentery.

PubMed

Han, Jing-Yan; Horie, Yoshinori; Miura, Soichiro; Akiba, Yasutada; Guo, Jun; Li, Dan; Fan, Jing-Yu; Liu, Yu-Ying; Hu, Bai-He; An, Li-Hua; Chang, Xin; Xu, Man; Guo, De-An; Sun, Kai; Yang, Ji-Ying; Fang, Shu-Ping; Xian, Ming-Ji; Kizaki, Masahiro; Nagata, Hiroshi; Hibi, Toshifumi

2007-07-14

To investigate the effect of compound Danshen injection on lipopolysaccharide (LPS)-induced rat mesenteric microcirculatory dysfunctions and the underlying possible mechanism by an inverted intravital microscope and high-speed video camera system. LPS was continuously infused through the jugular artery of male Wistar rats at the dose of 2 mg/kg per hour. Changes in mesenteric microcirculation, such as diameters of arterioles and venules, velocity of RBCs in venules, leukocyte rolling, adhesion and emigration, free radicals released from post-capillary venules, FITC-albumin leakage and mast cell degranulation, were observed through an inverted intravital microscope assisted with CCD camera and SIT camera. Meanwhile, the expression of adhesion molecules CD11b/CD18 and the production of free radical in neutrophils, and the expression of intercellular adhesion molecule 1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs) were quantified by flow cytometry (FACS) in vitro. The continuous infusion with LPS resulted in a number of responses in microcirculation, including a significant increase in the positive region of venule stained with Monastral blue B, rolling and adhesion of leukocytes, production of oxygen radical in venular wall, albumin efflux and enhanced mast cell degranulation in vivo, all of which, except for the leukocyte rolling, were attenuated by the treatment with compound Danshen injection. Experiments performed in vitro further revealed that the expression of CD11b/CD18 and the production of oxygen free radical in neutrophils, and the expression of ICAM-1 in HUVECs were increased by exposure to LPS, and they were attenuated by compound Danshen injection. These results suggest that compound Danshen injection is an efficient drug with multi-targeting potential for improving the microcirculatory disturbance.
Expression of the nifH Gene of a Herbaspirillum Endophyte in Wild Rice Species: Daily Rhythm during the Light-Dark Cycle

PubMed Central

You, Mu; Nishiguchi, Tomohiro; Saito, Asami; Isawa, Tsuyoshi; Mitsui, Hisayuki; Minamisawa, Kiwamu

2005-01-01

The expression of nitrogenase genes of Herbaspirillum sp. B501 associated in shoot (leaf and stem) of wild rice, Oryza officinalis, was studied by means of reverse transcription-PCR (RT-PCR) targeted at the nifH gene. RT-PCR analyses indicate that nifH transcript was detected exclusively from nitrogen-fixing cells of gfp-tagged strain B501gfp1 in both free-living and endophytic states by using a constitutive gfp gene transcript as a positive control. Transcription of nifH and nitrogen fixation in free-living cells were induced maximally at a 2% O2 concentration and repressed in free air (21% O2). nifH transcription was monitored in the endophytic cells by using total RNA extracted from B501gfp1-inoculated wild rice plants during daily light-dark cycles. The level of nifH transcription in planta varied dramatically, with a maximum during the light period. Moreover, the light radiation enhanced nifH expression even in free-living cells grown in culture. These results suggest that in planta nitrogen fixation by the endophyte shows a daily rhythm determined by the plant's light environment. PMID:16332801
Arachidonic acid can function as a signaling modulator by activating the TRPM5 cation channel in taste receptor cells.

PubMed

Oike, Hideaki; Wakamori, Minoru; Mori, Yasuo; Nakanishi, Hiroki; Taguchi, Ryo; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

2006-09-01

Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cbeta2, the TRPM5 channel is activated. Intracellular Ca(2+) is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A(2) (PLA(2)-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA(2)-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 microM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.
Human dental pulp stem cells cultured in serum-free supplemented medium

PubMed Central

Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

2013-01-01

Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs. PMID:24376422
Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells

PubMed Central

Young, J M; McNeilly, A S

2012-01-01

Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1–100 ng/ml) suppressed androstenedione production. Inhibin (1–100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells. PMID:22082494
An Organotypic 3D Assay for Primary Human Mammary Epithelial Cells that Recapitulates Branching Morphogenesis.

PubMed

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

2017-01-01

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

PubMed Central

Saubi, Narcís; Gea-Mallorquí, Ester; Ferrer, Pau; Hurtado, Carmen; Sánchez-Úbeda, Sara; Eto, Yoshiki; Gatell, Josep M; Hanke, Tomáš; Joseph, Joan

2014-01-01

In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)–mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice–compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on “double” auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth. PMID:26015961
Mannan oligosaccharide requires functional ETC and TLR for biological radiation protection to normal cells.

PubMed

Sanguri, Sweta; Gupta, Damodar

2018-06-27

Low LET Ionizing radiation is known to alter intracellular redox balance by inducing free radical generation, which may cause oxidative modification of various cellular biomolecules. The extent of biomolecule-modifications/ damages and changes in vital processes (viz. cellular homeostasis, inter-/intra-cellular signaling, mitochondrial physiology/dynamics antioxidant defence systems) are crucial which in turn determine fate of cells. In the present study, we expended TLR expressing (normal/ transformed) and TLR null cells; and we have shown that mannan pretreatment in TLR expressing normal cells offers survival advantage against lethal doses of ionizing radiation. On the contrary, mannan pretreatment does not offer any protection against radiation to TLR null cells, NKE ρ° cells and transformed cells. In normal cells, abrupt decrease in mitochondrial membrane potential and endogenous ROS levels occurs following treatment with mannan. We intend to irradiate mannan-pretreated cells at a specific stage of perturbed mitochondrial functioning and ROS levels to comprehend if mannan pretreatment offers any survival advantage against radiation exposure to cells. Interestingly, pre-irradiation treatment of cells with mannan activates NFκB, p38 and JNK, alters mitochondrial physiology, increases expression of Cu/ZnSOD and MnSOD, minimizes oxidation of mitochondrial phospholipids and offers survival advantage in comparison to irradiated group, in TLR expressing normal cells. The study demonstrates that TLR and mitochondrial ETC functions are inevitable in radio-protective efficacy exhibited by mannan.
Direct cell-cell contact activates SigM to express the ESX-4 secretion system in Mycobacterium smegmatis.

PubMed

Clark, Ryan R; Judd, Julius; Lasek-Nesselquist, Erica; Montgomery, Sarah A; Hoffmann, Jennifer G; Derbyshire, Keith M; Gray, Todd A

2018-06-25

Conjugal cell-cell contact between strains of Mycobacterium smegmatis induces the esxUT transcript, which encodes the putative primary substrates of the ESAT-6 secretion system 4 (ESX-4) secretion system. This recipient response was required for conjugal transfer of chromosomal DNA from the donor strain. Here we show that the extracytoplasmic σ factor, SigM, is a cell contact-dependent activator of ESX-4 expression and is required for conjugal transfer of DNA in the recipient strain. The SigM regulon includes genes outside the seven-gene core esx4 locus that we show are also required for conjugation, and we show that some of these SigM-induced proteins likely function through ESX-4. A fluorescent reporter revealed that SigM is specifically activated in recipient cells in direct contact with donor cells. Coculture RNA-seq experiments indicated that SigM regulon induction occurred early and before transconjugants are detected. This work supports a model wherein donor contact with the recipient cell surface inactivates the transmembrane anti-SigM, thereby releasing SigM. Free SigM induces an extended ESX-4 secretion system, resulting in changes that facilitate chromosomal transfer. The contact-dependent inactivation of an extracytoplasmic σ-factor that tightly controls ESX-4 activity suggests a mechanism dedicated to detect, and appropriately respond to, external stimuli from mycobacteria.
Mediation of glucolipotoxicity in INS-1 rat insulinoma cells by small heterodimer partner interacting leucine zipper protein (SMILE).

PubMed

Lee, Kyeong-Min; Seo, Ye Jin; Kim, Mi-Kyung; Seo, Hyun-Ae; Jeong, Ji-Yun; Choi, Hueng-Sik; Lee, In-Kyu; Park, Keun-Gyu

2012-03-23

Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.
A Graphene-enhanced imaging of microRNA with enzyme-free signal amplification of catalyzed hairpin assembly in living cells.

PubMed

Liu, Haiyun; Tian, Tian; Ji, Dandan; Ren, Na; Ge, Shenguang; Yan, Mei; Yu, Jinghua

2016-11-15

In situ imaging of miRNA in living cells could help us to monitor the miRNA expression in real time and obtain accurate information for studying miRNA related bioprocesses and disease. Given the low-level expression of miRNA, amplification strategies for intracellular miRNA are imperative. Here, we propose an amplification strategy with a non-destructive enzyme-free manner in living cells using catalyzed hairpin assembly (CHA) based on graphene oxide (GO) for cellular miRNA imaging. The enzyme-free CHA exhibits stringent recognition and excellent signal amplification of miRNA in the living cells. GO is a good candidate as a fluorescence quencher and cellular carrier. Taking the advantages of the CHA and GO, we can monitor the miRNA at low level in living cells with a simple, sensitive and real-time manner. Finally, imaging of miRNAs in the different expression cells is realized. The novel method could supply an effective tool to visualize intracellular low-level miRNAs and help us to further understand the role of miRNAs in cellular processes. Copyright © 2016 Elsevier B.V. All rights reserved.
High-Throughput Microfluidic Labyrinth for the Label-free Isolation of Circulating Tumor Cells.

PubMed

Lin, Eric; Rivera-Báez, Lianette; Fouladdel, Shamileh; Yoon, Hyeun Joong; Guthrie, Stephanie; Wieger, Jacob; Deol, Yadwinder; Keller, Evan; Sahai, Vaibhav; Simeone, Diane M; Burness, Monika L; Azizi, Ebrahim; Wicha, Max S; Nagrath, Sunitha

2017-09-27

We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations. Copyright © 2017 Elsevier Inc. All rights reserved.

Prognostic value of proliferating cell nuclear antigen in parotid gland cancer.

PubMed

Stenner, Markus; Demgensky, Ariane; Molls, Christoph; Hardt, Aline; Luers, Jan C; Grosheva, Maria; Huebbers, Christian U; Klussmann, Jens P

2012-04-01

Although cell proliferation is related to tumour aggressiveness and prognosis, there are few studies describing the expression of proliferative markers in salivary gland cancer. Our aim was to assess the long-term prognostic value of the proliferating cell nuclear antigen (PCNA) in a large group of histologically different salivary gland cancers. We analysed the expression of PCNA in 159 patients with parotid gland cancer by means of immunohistochemistry. The mean follow-up time was 56.6 months. A high expression of PCNA showed a significant correlation to the patients' pathological lymph node stage (p = 0.004). A high PCNA expression significantly indicated a poor 5-year disease-free (p = 0.046) and overall survival rate (p = 0.018). The PCNA expression was the only prognostic factor for a worse 5-year disease-free and overall survival in acinic cell carcinomas (p = 0.004, p = 0.022). The correlation between PCNA expression and survival probabilities of salivary gland cancer might make proliferation markers helpful tools in patient follow-up, prognosis and targeted therapy in salivary gland cancer in future.
Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses.

PubMed

Chen, Ping; Hübner, Wolfgang; Spinelli, Matthew A; Chen, Benjamin K

2007-11-01

Cell-free human immunodeficiency virus type 1 (HIV-1) can initiate infections, but contact between infected and uninfected T cells can enhance viral spread through intercellular structures called virological synapses (VS). The relative contribution of VS to cell-free viral transfer has not been carefully measured. Using an ultrasensitive, fluorescent virus transfer assay, we estimate that when VS between HIV-expressing Jurkat T cells and primary CD4(+) T cells are formed, cell-associated transfer of virus is 18,000-fold more efficient than uptake of cell-free virus. Furthermore, in contrast to cell-free virus uptake, the VS deposits virus rapidly into focal, trypsin-resistant compartments in target T cells. This massive virus internalization requires Env-CD4 receptor interactions but is resistant to inhibition by patient-derived neutralizing antisera that inhibit homologous cell-free virus. Deleting the Env cytoplasmic tail does not abrogate VS-mediated transfer, but it renders the VS sensitive to neutralizing antibodies, suggesting that the tail limits exposure of VS-neutralizing epitopes on the surface of infected cells. Dynamic live imaging of the VS reveals that HIV-expressing cells are polarized and make sustained, Env-dependent contacts with target cells through uropod-like structures. The polarized T-cell morphology, Env-CD4 coordinated adhesion, and viral transfer from HIV-infected to uninfected cells suggest that VS allows HIV-1 to evade antibody neutralization and to disseminate efficiently. Future studies will discern to what extent this massive viral transfer contributes to productive infection or viral dissemination through the migration of virus-carrying T cells.
Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.

PubMed

Marshall, Ryan; Maxwell, Colin S; Collins, Scott P; Jacobsen, Thomas; Luo, Michelle L; Begemann, Matthew B; Gray, Benjamin N; January, Emma; Singer, Anna; He, Yonghua; Beisel, Chase L; Noireaux, Vincent

2018-01-04

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses. Copyright © 2017 Elsevier Inc. All rights reserved.
A novel role of HLA class I in the pathology of medulloblastoma.

PubMed

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-07-12

MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. We investigated expression of four essential components of MHC class I (heavy chain, beta2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of beta2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or beta2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.
A novel role of HLA class I in the pathology of medulloblastoma

PubMed Central

Smith, Courtney; Santi, Mariarita; Rajan, Bhargavi; Rushing, Elisabeth J; Choi, Mi Rim; Rood, Brian R; Cornelison, Robert; MacDonald, Tobey J; Vukmanovic, Stanislav

2009-01-01

Background MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. Methods We investigated expression of four essential components of MHC class I (heavy chain, β2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. Results The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of β2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. Conclusion MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or β2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility. PMID:19594892
Method To Identify Specific Inhibiutors Of Imp Dehydrogenase

DOEpatents

Collart, Frank R.; Huberman, Eliezer

2000-11-28

This invention relates to methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, IMP dehydrogenase (IMPDH). IMPDH is an essential enzyme found in all free-living organisms from humans to bacteria and is an important therapeutic target. The invention allows the identification of specific inhibitors of any IMPDH enzyme which can be expressed in a functional form in a recombinant host cell. A variety of eukaryotic or prokaryotic host systems commonly used for the expression of recombinant proteins are suitable for the practice of the invention. The methods are amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods such as antisense molecule production. Utilization of exogenous guanosine as a control component of the methods allows for the identification of inhibitors specific for IMPDH rather than other causes of decreased cell proliferation.
Niemann-Pick Type C2 Protein Regulates Free Cholesterol Accumulation and Influences Hepatic Stellate Cell Proliferation and Mitochondrial Respiration Function.

PubMed

Wang, Yuan-Hsi; Twu, Yuh-Ching; Wang, Chung-Kwe; Lin, Fu-Zhen; Lee, Chun-Ya; Liao, Yi-Jen

2018-06-05

Liver fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. A high-cholesterol diet is associated with liver fibrosis via the accumulation of free cholesterol in hepatic stellate cells (HSCs). Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular free cholesterol homeostasis via direct binding with free cholesterol. Previously, we reported that NPC2 was downregulated in liver cirrhosis tissues. Loss of NPC2 enhanced the accumulation of free cholesterol in HSCs and made them more susceptible to transforming growth factor (TGF)-β1. In this study, we showed that knockdown of NPC2 resulted in marked increases in platelet-derived growth factor BB (PDGF-BB)-induced HSC proliferation through enhanced extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK), and protein kinase B (AKT) phosphorylation. In contrast, NPC2 overexpression decreased PDGF-BB-induced cell proliferation by inhibiting p38, JNK, and AKT phosphorylation. Although NPC2 expression did not affect caspase-related apoptosis, the autophagy marker light chain 3β (LC3B) was decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs. The mitochondrial respiration functions (such as oxygen consumption rate, ATP production, and maximal respiratory capacity) were decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs, while NPC2-overexpressed cells remained normal. In addition, NPC2 expression did not affect the susceptibility of HSCs to lipopolysaccharides (LPS), and U18666A treatment induced free cholesterol accumulation, which enhanced LPS-induced Toll-like receptor 4 (TLR4), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation, interleukin (IL)-1 and IL-6 expression. Our study demonstrated that NPC2-mediated free cholesterol homeostasis controls HSC proliferation and mitochondrial function.
Involvement of gut microbiota in association between GLP-1/GLP-1 receptor expression and gastrointestinal motility.

PubMed

Yang, Mo; Fukui, Hirokazu; Eda, Hirotsugu; Xu, Xin; Kitayama, Yoshitaka; Hara, Ken; Kodani, Mio; Tomita, Toshihiko; Oshima, Tadayuki; Watari, Jiro; Miwa, Hiroto

2017-04-01

The microbiota in the gut is known to play a pivotal role in host physiology by interacting with the immune and neuroendocrine systems in gastrointestinal (GI) tissues. Glucagon-like peptide 1 (GLP-1), a gut hormone, is involved in metabolism as well as GI motility. We examined how gut microbiota affects the link between GLP-1/GLP-1 receptor (GLP-1R) expression and motility of the GI tract. Germ-free (GF) mice (6 wk old) were orally administered a fecal bacterial suspension prepared from specific pathogen-free (SPF) mice, and then after fecal transplantation (FT) GI tissues were obtained from the GF mice at various time points. The expression of GLP-1 and its receptor was examined by immunohistochemistry, and gastrointestinal transit time (GITT) was measured by administration of carmine red solution. GLP-1 was expressed in endocrine cells in the colonic mucosa, and GLP-1R was expressed in myenteric neural cells throughout the GI wall. GLP-1R-positive cells throughout the GI wall were significantly fewer in GF mice with FT than in GF mice without gut microbiota reconstitution. GITT was significantly shorter in GF mice with FT than in control GF mice without FT and correlated with the number of GLP-1R-positive cells throughout the GI wall. GITT was significantly longer in GF control mice than in SPF mice. When those mice were treated with GLP-1 agonist extendin4, GITT was significantly longer in the GF mice. The gut microbiota may accelerate or at least modify GI motility while suppressing GLP-1R expression in myenteric neural cells throughout the GI tract. NEW & NOTEWORTHY The gut microbiota has been intensively studied, because it plays a pivotal role in various aspects of host physiology. On the other hand, glucagon-like peptide 1 (GLP-1) plays important roles in metabolism as well as gastrointestinal motility. In the present study, we have suggested that the gut microbiota accelerates gastrointestinal motility while suppressing the expression of GLP-1 receptor in myenteric neural cells throughout the gastrointestinal tract. We believe that this article is very timely and suggestive work. Copyright © 2017 the American Physiological Society.
NIPA-like domain containing 1 is a novel tumor-promoting factor in oral squamous cell carcinoma.

PubMed

Sasahira, Tomonori; Nishiguchi, Yukiko; Kurihara-Shimomura, Miyako; Nakashima, Chie; Kuniyasu, Hiroki; Kirita, Tadaaki

2018-05-01

In our previous global gene expression analysis, we identified NIPA-like domain containing 1 (NIPAL1), which encodes a magnesium transporter, as one of the most overexpressed genes in recurrent oral squamous cell carcinoma (OSCC). Although has been NIPAL1 linked with gout pathogenesis, little is known about its expression and function in human malignancies. In this study, we examined NIPAL1 expression in 192 cases of OSCC by immunohistochemistry and performed a functional analysis of human OSCC cells. NIPAL1 immunostaining was observed in 39 of 192 OSCC patients (20.3%). NIPAL1 expression correlated significantly with cancer cell intravsation (P = 0.0062), as well as with poorer disease-free survival in a Kaplan-Meier analysis (P < 0.0001). Moreover, a multivariate Cox proportional hazards model analysis revealed that NIPAL1 expression was an independent predictor of disease-free survival in OSCC (P < 0.0001). In a functional analysis, NIPAL1 regulated the growth and adhesion of OSCC tumor cells and endothelial cells. Our findings suggest that NIPAL1 might be a novel factor promoting OSCC tumorigenesis, as well as a useful molecular marker of OSCC.
Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line.

PubMed

Rasmussen, B; Davis, R; Thomas, J; Reddy, P

1998-11-01

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day.
Ceruloplasmin expression by human peripheral blood lymphocytes: a new link between immunity and iron metabolism.

PubMed

Banha, João; Marques, Liliana; Oliveira, Rita; Martins, Maria de Fátima; Paixão, Eleonora; Pereira, Dina; Malhó, Rui; Penque, Deborah; Costa, Luciana

2008-02-01

Ceruloplasmin (CP) is a multicopper oxidase involved in the acute phase reaction to stress. Although the physiological role of CP is uncertain, its role in iron (Fe) homeostasis and protection against free radical-initiated cell injury has been widely documented. Previous studies showed the existence of two molecular isoforms of CP: secreted CP (sCP) and a membrane glycosylphosphatidylinositol (GPI)-anchored form of CP (GPI-CP). sCP is produced mainly by the liver and is abundant in human serum whereas GPI-CP is expressed in mammalian astrocytes, rat leptomeningeal cells, and Sertolli cells. Herein, we show using RT-PCR that human peripheral blood lymphocytes (huPBL) constitutively express the transcripts for both CP molecular isoforms previously reported. Also, expression of CP in huPBL is demonstrated by immunofluorescence with confocal microscopy and flow cytometry analysis using cells isolated from healthy blood donors with normal Fe status. Importantly, the results obtained show that natural killer cells have a significantly higher CP expression compared to all other major lymphocyte subsets. In this context, the involvement of lymphocyte-derived CP on host defense processes via its anti/prooxidant properties is proposed, giving further support for a close functional interaction between the immune system and the Fe metabolism.
Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

PubMed

Cao, Ming-Xia; Huang, Jian-Qiu; Yao, Quan-Hong; Liu, Sheng-Jun; Wang, Cheng-Long; Wei, Zhi-Ming

2006-01-01

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.
Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).

PubMed

He, M; Taussig, M J

2001-08-01

We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.
[Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma].

PubMed

Ren, K; Qian, D; Wang, Y W; Pang, Q S; Zhang, W C; Yuan, Z Y; Wang, P

2018-01-23

Objective: To investigate the clinical significance of NS1-BP expression in patients with esophageal squamous cell carcinoma (ESCC), and to study the roles of NS1-BP in proliferation and apoptosis of ESCC cells. Methods: A total of 98 tumor tissues and 30 adjacent normal tissues from 98 ESCC patients were used as study group and control group, and these samples were collected in Sun Yat-Sen University Cancer Center between 2002 and 2008. In addition, 46 ESCC tissues which were collected in Cancer Institute and Hospital of Tianjin Medical University were used as validation group. Expression of mucosal NS1-BP was detected by immunohistochemistry. Kaplan-Meier curve and log-rank test were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors. Furthermore, NS1-BP was over expressed or knocked down in ESCC cells by transient transfection. Protein levels of c-Myc were detected by western blot. Cell viability and apoptosis was analyzed by MTT assay and flow cytometry. Results: Among all of tested samples, NS1-BP were down-regulated in 9 out of 30 non-tumorous normal esophageal tissues (30.0%) and 85 out of 144 ESCC tissues (59.0%), respectively, showing a statistically significant difference ( P =0.012). In the study group, three-year disease-free survival rate of NS1-BP high expression group (53.2%) was significantly higher than that of NS1-BP low expression group (27.6%; P =0.009). In the validation group, the three-year disease-free survival rates were 57.8% and 25.5% in NS1-BP high and low levels groups, respectively, showing a similar results ( P =0.016). Importantly, multivariate analyses showed that low expression of NS1-BP was an independent predictor for chemoradiotherapy sensitivity and shorter disease-free survival time in ESCC patients( P <0.05 for all). Furthermore, overexpressed NS1-BP in TE-1 cells repressed c-Myc expression, inhibited cell proliferation and promoted apoptosis. In contrast, knockdown NS1-BP in KYSE510 cells induced c-Myc expression, increased cell proliferation and repressed apoptosis. Conclusions: NS1-BP is an independent favorable prognostic factor in ESCC. It inhibits cell proliferation and enhances cell apoptosis via repressing c-Myc. Targeting NS1-BP may be a new therapeutic strategy for ESCC patients.
Cytoplasmic expression of Twist1, an EMT-related transcription factor, is associated with higher grades renal cell carcinomas and worse progression-free survival in clear cell renal cell carcinoma.

PubMed

Rasti, Arezoo; Madjd, Zahra; Abolhasani, Maryam; Mehrazma, Mitra; Janani, Leila; Saeednejad Zanjani, Leili; Asgari, Mojgan

2018-05-01

Twist1 is a key transcription factor, which confers tumor cells with cancer stem cell (CSC)-like characteristics and enhances epithelial-mesenchymal transition in pathological conditions including tumor malignancy and metastasis. This study aimed to evaluate the expression patterns and clinical significance of Twist1 in renal cell carcinoma (RCC). The cytoplasmic and nuclear expression of Twist1 were examined in 252 well-defined renal tumor tissues, including 173 (68.7%) clear cell renal cell carcinomas (ccRCC), 45 (17.9%) papillary renal cell carcinomas (pRCC) and 34 (13.5%) chromophobe renal cell carcinoma, by immunohistochemistry on a tissue microarray. The association between expression of this marker and clinicopathologic parameters and survival outcomes were then analyzed. Twist1 was mainly localized to the cytoplasm of tumor cells (98.8%). Increased cytoplasmic expression of Twist1 was associated with higher grade tumors (P = 0.045), renal vein invasion (P = 0.031) and microvascular invasion (P = 0.044) in RCC. It was positively correlated with higher grade tumors (P = 0.026), shorter progression-free survival time (P = 0.027) in patients with ccRCC, and also with higher stage in pRCC patients (P = 0.036). Significantly higher cytoplasmic expression levels of Twist1 were found in ccRCC and pRCC subtypes, due to their more aggressive tumor behavior. Increased cytoplasmic expression of Twist1 had a critical role in worse prognosis in ccRCC. These findings suggest that cytoplasmic, rather than nuclear expression of Twist1 can be considered as a prognostic and therapeutic marker for targeted therapy of RCC, especially for ccRCC patients.
Intratumoral immune cells expressing PD-1/PD-L1 and their prognostic implications in cancer: a meta-analysis.

PubMed

Kim, Younghoon; Wen, Xianyu; Cho, Nam Yun; Kang, Gyeong Hoon

2018-05-01

The prognostic value of immune cells expressing programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) in cancer are controversial, and the potential differential impact of using tissue microarrays and whole tissue sections to assess the positivity of immune cells has not been addressed. The current study included 30 eligible studies with 7251 patients that evaluated the relationship between tumor-infiltrating lymphocytes expressing PD-1/PD-L1 and overall survival and disease-free survival, or progression-free survival. Subgroup analysis was based on the tissue type of cancer and the type of tissue sampling (tissue microarray or whole tissue section). In the meta-analysis, PD-1-positive and PD-L1-positive tumor-infiltrating lymphocytes had a positive effect on disease-free survival or progression-free survival (hazard ratio [HR] 0.732; 95% confidence interval [CI] 0.565, 0.947; and HR 0.727; 95% CI 0.584, 0.905, respectively). PD-L1-positive tumor-infiltrating lymphocytes had a positive impact on overall survival in studies using tissue microarray (HR 0.586; 95% CI 0.476, 0.721), but had a poor impact when only whole tissue sections were considered (HR 1.558; 95% CI 1.232, 1.969). Lung cancer was associated with good overall survival and disease-free survival (HR 0.639; 95% CI 0.491, 0.831; and HR 0.693; 95% CI 0.538, 0.891, respectively) for PD-1-positive tumor-infiltrating lymphocytes, and colorectal cancer showed favorable disease-free survival (HR 0.471; 95% CI 0.308, 0.722) for PD-L1-positive tumor-infiltrating lymphocytes. Immune cells expressing PD-1 and PD-L1 within tumors are associated with the prognosis. However, the correlation may vary among different tumor types and by the type of tissue sampling used for the assessment.
Ghrelin inhibits proliferation and increases T-type Ca{sup 2+} channel expression in PC-3 human prostate carcinoma cells

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

Diaz-Lezama, Nundehui; Hernandez-Elvira, Mariana; Sandoval, Alejandro

Research highlights: {yields} Ghrelin decreases prostate carcinoma PC-3 cells proliferation. {yields} Ghrelin favors apoptosis in PC-3 cells. {yields} Ghrelin increase in intracellular free Ca{sup 2+} levels in PC-3 cells. {yields} Grelin up-regulates expression of T-type Ca{sup 2+} channels in PC-3 cells. {yields} PC-3 cells express T-channels of the Ca{sub V}3.1 and Ca{sub V}3.2 subtype. -- Abstract: Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating themore » cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca{sup 2+} levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca{sup 2+} channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca{sup 2+} channel expression.« less
Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

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

Belfort, Georges; Grimaldi, Joseph J.

2015-01-27

Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), andmore » (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the presence of glycocalyx filaments coating the luminal surface of our vasculature and represent a new class of synthetic membranes. They thus meet the requirements/scope of the Bimolecular Materials program, Materials Science and Engineering Div., Office of Science, US DOE.« less
Comparison of Synthetic Media Designed for Expansion of Adipose-Derived Mesenchymal Stromal Cells.

PubMed

Lensch, Michelle; Muise, Angela; White, Lisa; Badowski, Michael; Harris, David

2018-05-14

Mesenchymal stromal cells (MSCs) are multipotent cells that can differentiate into various cell types, such as osteoblasts, myocytes, and adipocytes. This characteristic makes the cells a useful tool in developing new therapies for a number of common maladies and diseases. The utilization of animal-derived growth serum, such as fetal bovine serum (FBS), for the expansion of MSCs has traditionally been used for cell culture. However, in clinical applications, animal-derived products present limitations and safety concerns for the recipient, as exposure to animal (xeno-) antigens and infectious agents is possible. Multiple synthetic, xeno-free media have been developed to combat these limitations of animal-derived growth serum and have the potential to be used in ex vivo MSC expansion for clinical use. The goal of this study was to determine if xeno-free media are adequate to significantly and efficiently expand MSCs derived from adipose tissue. MSCs were cultured in both standard FBS-containing as well as xeno-free media. The media were compared for cell yield, viability, and phenotypic expression via flow cytometry and directed differentiation. The xeno-free media that were tested were StemMACS MSC Expansion Media (Miltenyi Biotec, Bergisch Gladbach, Germany), PLTMax Human Platelet Lysate (Sigma-Aldrich, St. Louis, MO, USA), and MesenCult-hPL media (Stemcell Technologies, Vancouver, BC, Canada). All xeno-free media showed promise as a feasible replacement for animal-derived growth serums. The xeno-free media expanded MSCs more quickly than the FBS-containing medium and also showed great similarity in cell viability and phenotypic expression. In fact, each xeno-free media produced a greater viable cell yield than the standard FBS-containing medium.
Clinical evaluation of a novel microfluidic device for epitope-independent enrichment of circulating tumour cells in patients with small cell lung cancer.

PubMed

Chudziak, Jakub; Burt, Deborah J; Mohan, Sumitra; Rothwell, Dominic G; Mesquita, Bárbara; Antonello, Jenny; Dalby, Suzanne; Ayub, Mahmood; Priest, Lynsey; Carter, Louise; Krebs, Matthew G; Blackhall, Fiona; Dive, Caroline; Brady, Ged

2016-01-21

Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.

CD44 increases the efficiency of distant metastasis of breast cancer

PubMed Central

McFarlane, Suzanne; Coulter, Jonathan A.; Tibbits, Paul; O'Grady, Anthony; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; McCarthy, Helen O.; Young, Leonie S.; Kay, Elaine W.; Isacke, Clare M.; Waugh, David J.J.

2015-01-01

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients. PMID:25888636
The Combinational Use of CRISPR/Cas9 and Targeted Toxin Technology Enables Efficient Isolation of Bi-Allelic Knockout Non-Human Mammalian Clones.

PubMed

Watanabe, Satoshi; Sakurai, Takayuki; Nakamura, Shingo; Miyoshi, Kazuchika; Sato, Masahiro

2018-04-04

Recent advances in genome editing systems such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) have facilitated genomic modification in mammalian cells. However, most systems employ transient treatment with selective drugs such as puromycin to obtain the desired genome-edited cells, which often allows some untransfected cells to survive and decreases the efficiency of generating genome-edited cells. Here, we developed a novel targeted toxin-based drug-free selection system for the enrichment of genome-edited cells. Cells were transfected with three expression vectors, each of which carries a guide RNA (gRNA), humanized Cas9 ( hCas9 ) gene, or Clostridium perfringens -derived endo-β-galactosidase C ( EndoGalC ) gene. Once EndoGalC is expressed in a cell, it digests the cell-surface α-Gal epitope, which is specifically recognized by BS-I-B₄ lectin (IB4). Three days after transfection, these cells were treated with cytotoxin saporin-conjugated IB4 (IB4SAP) for 30 min at 37 °C prior to cultivation in a normal medium. Untransfected cells and those weakly expressing EndoGalC will die due to the internalization of saporin. Cells transiently expressing EndoGalC strongly survive, and some of these surviving clones are expected to be genome-edited bi-allelic knockout (KO) clones due to their strong co-expression of gRNA and hCas9. When porcine α-1,3-galactosyltransferase gene, which can synthesize the α-Gal epitope, was attempted to be knocked out, 16.7% and 36.7% of the surviving clones were bi-allelic and mono-allelic knockout (KO) cells, respectively, which was in contrast to the isolation of clones in the absence of IB4SAP treatment. Namely, 0% and 13.3% of the resulting clones were bi-allelic and mono-allelic KO cells, respectively. A similar tendency was seen when other target genes such as DiGeorge syndrome critical region gene 2 and transforming growth factor-β receptor type 1 gene were targeted to be knocked out. Our results indicate that a combination of the CRISPR/Cas9 system and targeted toxin technology using IB4SAP allows efficient enrichment of genome-edited clones, particularly bi-allelic KO clones.
A chromosomally encoded T7 RNA polymerase-dependent gene expression system for Corynebacterium glutamicum: construction and comparative evaluation at the single-cell level

PubMed Central

Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael

2015-01-01

Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum. PMID:25488698
Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1)-Harnessed SAINT-Based Lipid Carrier System

PubMed Central

Visweswaran, Ganesh Ram R.; Gholizadeh, Shima; Ruiters, Marcel H. J.; Molema, Grietje; Kok, Robbert J.; Kamps, Jan. A. A. M.

2015-01-01

Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes. PMID:26407295
Mast cell heterogeneity and anti-inflammatory annexin A1 expression in leprosy skin lesions.

PubMed

Costa, Maurício B; Mimura, Kallyne K O; Freitas, Aline A; Hungria, Emerith M; Sousa, Ana Lúcia O M; Oliani, Sonia M; Stefani, Mariane M A

2018-03-29

Mast cells (MCs) have important immunoregulatory roles in skin inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory protein that can be expressed by mast cells, neutrophils, eosinophils, monocytes, epithelial and T cells. This study investigated MCs heterogeneity and ANXA1 expression in human dermatoses with special emphasis in leprosy. Sixty one skin biopsies from 2 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 type 1 reaction/T1R, 11 type 2 reaction/T2R); 21 patients with other dermatoses. Tryptase/try+ and chymase/chy + phenotypic markers and toluidine blue stained intact/degranulated MC counts/mm 2 were evaluated. Try + /chy + MCs and ANXA1 were identified by streptavidin-biotin-peroxidase immunostaining and density was reported. In leprosy, degranulated MCs outnumbered intact ones regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), leprosy reactions (reactional/reaction-free) and type of reaction (T1R/T2R). Compared to other dermatoses, leprosy skin lesions showed lower numbers of degranulated and intact MCs. Try + MCs outnumbered chy + in leprosy lesions (reaction-free/reactional, particularly in T2R), but not in other dermatoses. Compared to other dermatoses, ANXA1 expression, which is also expressed in mast cells, was higher in the epidermis of leprosy skin lesions, independently of reactional episode. In leprosy, higher MC degranulation and differential expression of try + /chy + subsets independent of leprosy type and reaction suggest that the Mycobacterium leprae infection itself dictates the inflammatory MCs activation in skin lesions. Higher expression of ANXA1 in leprosy suggests its potential anti-inflammatory role to maintain homeostasis preventing tissue and nerve damage. Copyright © 2018 Elsevier Ltd. All rights reserved.
Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems

PubMed Central

2014-01-01

RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo. PMID:24621257
MicroRNA-34c-5p is related to recurrence in laryngeal squamous cell carcinoma.

PubMed

Re, Massimo; Çeka, Artan; Rubini, Corrado; Ferrante, Luigi; Zizzi, Antonio; Gioacchini, Federico M; Tulli, Michele; Spazzafumo, Liana; Sellari-Franceschini, Stefano; Procopio, Antonio D; Olivieri, Fabiola

2015-09-01

Altered microRNA expression has been found in many cancer types, including laryngeal squamous cell carcinoma (LSCC). We investigated the association of LSCC-related miR-34c-5p with disease-free survival and overall survival. Retrospective cohort study. Expression levels of miR-34c-5p were detected in 90 LSCC formalin-fixed paraffin-embedded tissues by reverse-transcription quantitative polymerase chain reaction. Overall survival and disease-free survival were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using Cox proportional hazard analysis. A downregulation of miR-34c-5p expression significantly correlated with worse disease-free and overall survival. In the multivariate analysis, low miR-34c-5p expression was associated with an increased risk of recurrence. A downregulation of miR-34c-5p in LSCC is independently associated with unfavorable disease-free survival, suggesting that miR-34c-5p might be a promising marker for evaluating the risk of recurrences. NA. © 2015 The American Laryngological, Rhinological and Otological Society, Inc.
Direct quantitative detection for cell-free miR-155 in urine: a potential role in diagnosis and prognosis for non-muscle invasive bladder cancer.

PubMed

Zhang, Xin; Zhang, Yanli; Liu, Xinfeng; Fang, Aiju; Wang, Jinfeng; Yang, Yongmei; Wang, Lili; Du, Lutao; Wang, Chuanxin

2016-01-19

High recurrence rates of non-muscle invasive bladder cancer (NMIBC) in patients require lifelong testing and monitoring. The aim of this study is to develop a simplified RT-qPCR method (RT-qPCR-D) which directly quantifies cell-free miR-155 in urine without RNA extraction, and assess it as a potential tool in NMIBC detection. A pilot study including 60 urine samples was used to investigate the feasibility of RT-qPCR-D in detecting cell-free miR-155. Then, miR-155 levels were quantified in a large independent cohort of urine from 162 NIMBC patients, 76 cystitis patients, and 86 healthy donors using the RT-qPCR-D method. Changes of cell-free miR-155 before and after operation were also analyzed in 32 NIMBC patients. In pilot study, we found a significant linear association between RT-qPCR and RT-qPCR-D in urinary miR-155 detection. Both methods showed cell-free miR-155 were significantly increased in NMIBC patients, and could reflect their expression in tissues. Then, the increased expression of cell-free miR-155 was successfully validated in 162 NIMBC patients when compared with cystitis patients and healthy donors. Moreover, it distinguished NMIBC patients from others with 80.2% sensitivity and 84.6% specificity, which was superior to urine cytology. Cell-free miR-155 correlated with NMIBC stage and grade, and was an independent factor for predicting recurrence and progression to muscle invasion. In addition, cell-free miR-155 was significantly decreased after NMIBC patients underwent transurethral bladder resection. In conclusion, detection of cell-free miR-155 in urine using RT-qPCR-D is a simple and noninvasive approach which may be used for NMIBC diagnosis and prognosis prediction.
Expression and in vitro regulation of integrins by normal human urothelial cells.

PubMed

Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

1995-08-01

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.
Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells

PubMed Central

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

2010-01-01

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a+CD42b+ platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b+ platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones. PMID:21098095
Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris.

PubMed

Sweat, J M; Johnson, C M; Marikar, Y; Gibbs, E P

2005-12-15

An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.
Doxorubicin-anti-carcinoembryonic antigen immunoconjugate activity in vitro.

PubMed

Richardson, V J; Ford, C H; Tsaltas, G; Gallant, M E

1989-04-01

An in vitro model consisting of a series of 11 human cancer cell lines with varying density of expression of membrane carcinoembryonic antigen (CEA) has been used to evaluate conjugates of doxorubicin (Adriamycin) covalently linked by a carbodiimide method to goat polyclonal antibodies and mouse monoclonal antibodies to CEA. Conjugates were produced which retained both antigen binding and drug cytotoxicity. IC50 values were determined for free drug, free drug mixed with unconjugated antibodies and for the immunoconjugates. Cell lines that were very sensitive to free drug (IC50 less than 100 ng/ml) were also found to be highly sensitive to conjugated drug and similarly cell lines resistant to drug (IC50 greater than 1,000 ng/ml) were also resistant to conjugated drug. Although there was no correlation between CEA expression and conjugates efficacy, competitive inhibition studies using autologous antibody to block conjugate binding to cells indicated immunoconjugates specificity for the CEA target.
Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

PubMed

Fogeron, Marie-Laure; Badillo, Aurélie; Jirasko, Vlastimil; Gouttenoire, Jérôme; Paul, David; Lancien, Loick; Moradpour, Darius; Bartenschlager, Ralf; Meier, Beat H; Penin, François; Böckmann, Anja

2015-01-01

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays. Copyright © 2014 Elsevier Inc. All rights reserved.
Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

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

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less
Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

DOE PAGES

Soranzo, Thomas; Martin, Donald K.; Lenormand, Jean -Luc; ...

2017-06-13

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for bothmore » direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.« less
Label-free screening of niche-to-niche variation in satellite stem cells using functionalized pores

NASA Astrophysics Data System (ADS)

Chapman, Matthew R.; Balakrishnan, Karthik; Conboy, Michael J.; Mohanty, Swomitra; Jabart, Eric; Huang, Haiyan; Hack, James; Conboy, Irina M.; Sohn, Lydia L.

2012-02-01

Combinations of surface markers are currently used to identify muscle satellite cells. Using pores functionalized with specific antibodies and measuring the transit time of cells passing through these pores, we discovered remarkable heterogeneity in the expression of these markers in muscle (satellite) stem cells that reside in different single myofibers. Microniche-specific variation in stem cells of the same organ has not been previously described, as bulk analysis does not discriminate between separate myofibers or even separate hind-leg muscle groups. We found a significant population of Sca-1+ satellite cells that form myotubes, thereby demonstrating the myogenic potential of Sca-1+ cells, which are currently excluded in bulk sorting. Finally, using our label-free pore screening technique, we have been able to quantify directly surface expression of Notch1 without activation of the Notch pathway. We show for the first time Notch1-expression heterogeneity in unactivated satellite cells. The discovery of fiber-to-fiber variations prompts new research into the reasons for such diversity in muscle stem cells.
GPER-1 acts as a tumor suppressor in ovarian cancer

PubMed Central

2013-01-01

Background It is known that the new membrane-bound estrogen receptor GPER-1 acts suppressive in breast cancer cells and its expression decreases during disease progression. This study was conducted to evaluate the GPER-1 expression in ovarian cancer and its correlation with progression. Its function was tested in vitro in ovarian cancer cells. Patients and methods GPER-1 expression was analyzed by immunohistochemistry in 35 benign ovarian tumors, 35 tumors of low-malignant potential and in 124 ovarian cancers. GPER-1 expression was correlated to the prospectively evaluated disease-free survival of ovarian cancer patients. We also tested GPER-1 expression in ovarian cancer cells and the effect of GPER-1 stimulation on cell growth. Results GPER-1 expression was significantly lower in ovarian cancer tissue than in benign and low-malignant ovarian tumors. GPER-1 expression was observed in 83.1% of malignant tumors and was higher in early stage cancers and tumors with high histological differentiation. GPER-1 expression was associated with favourable clinical outcome. The difference in 2-year disease-free survival by GPER-1 expression was significant, 28.6% for GPER-1 negative and 59.2% for GPER-1 positive cases (p = 0.002). GPER-1 expression was observed in SKOV-3 and OVCAR-3 ovarian cancer cell lines. G-1, a selective GPER-1 agonist, suppressed proliferation of the two cell types via inhibition of cell cycle progression in G2/M phase and stimulation of caspase-dependent apoptosis. The blockade in G2/M phase was associated with increased expression of cyclin B1 and Cdc2 and phosphorylation of histone 3. Conclusion GPER-1 emerges as a new tumor suppressor with unsuspected therapeutic potential for ovarian cancer. PMID:23849542
A real-time control system of gene expression using ligand-bound nucleic acid aptamer for metabolic engineering.

PubMed

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

2017-07-01

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

PubMed

Saadeldin, Islam M; Swelum, Ayman Abdel-Aziz; Elsafadi, Mona; Moumen, Abdullah F; Alzahrani, Faisal A; Mahmood, Amer; Alfayez, Musaad; Alowaimer, Abdullah N

2017-09-01

We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to ∼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels. Copyright © 2017 Elsevier Ltd. All rights reserved.
The Impact of PD-L1 Expression in Patients with Metastatic GEP-NETs

PubMed Central

Kim, Seung Tae; Ha, Sang Yun; Lee, Sujin; Ahn, Soomin; Lee, Jeeyun; Park, Se Hoon; Park, Joon Oh; Lim, Ho Yeong; Kang, Won Ki; Kim, Kyoung-Mee; Park, Young Suk

2016-01-01

Programmed death-ligand 1 (PD-L1), which is expressed on many cancer cells, interacts with PD1 expressed on the surface of T cells, inhibiting the T cells and blocking the antitumor immune response. Expression of PD-L1 in gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has not been studied. We investigated the impact of PD-L1 expression in 32 patients with metastatic GEP-NET. The expression of PD-L1 was evaluated using an anti-PD-L1 immunohistochemistry (IHC) antibody optimized for staining of formalin-fixed paraffin-embedded (FFPE) tissue samples. The correlation between PD-L1 and clinicopathological data including survival and response to systemic treatments was analyzed. Primary sites were 24 foregut-derived GEP-NETs, including stomach (n=1), duodenum (n=2), biliary tract (n=7), and pancreas (n=14), and 8 hindgut-derived GEP-NETs of the distal colon and rectum. Among the 32 patients with metastatic GEP-NET analyzed in this study, 7 (21.9%) had expression of PD-L1 in tumor tissues. Expression of PD-L1 was significantly associated with high-grade WHO classification (grade 3) (p=0.008) but not with gender, primary site, and number of metastatic sites (p>0.05). The status of PD-L1 expression was statistically associated with progression-free survival (PFS) for first-line systemic treatment (p=0.047). Moreover, the status of PD-L1 expression could significantly predict overall survival (p=0.037). The expression of PD-L1 was associated with higher WHO tumor grade (grade 3) in metastatic GEP-NETs. PD-L1 expression had both predictive and prognostic value for survival of patients with metastatic GEP-NETs. PMID:26958083

Stimulation of cAMP signalling allows isolation of clonal pancreatic precursor cells from adult mouse pancreas.

PubMed

Yamamoto, T; Yamato, E; Taniguchi, H; Shimoda, M; Tashiro, F; Hosoi, M; Sato, T; Fujii, S; Miyazaki, J-I

2006-10-01

Duct cells of the pancreas are thought to include latent progenitors of islet endocrine cells that can be induced to differentiate by appropriate morphogens. Here we developed a method for isolating pancreatic ductal epithelial cells from adult mice that overcomes the shortcomings of previous methods. Pancreatic ductal cells were grown in serum-free DMEM/F12 medium in the presence of cholera toxin or 8-bromo-cyclic adenosine monophosphate, which is known to be an intracellular cAMP generator. Single cell cloning was performed by limiting dilution in serum-free medium. The isolated clonal cells expressed high levels of cytokeratin and Ipf1 (formerly known as Pdx-1). Adenovirus-mediated expression of ngn3 (also known as Neurog3) and Ptf1a in these cells induced expression of insulin and somatostatin, and of carboxypeptidase A, respectively. Furthermore, albumin production was induced by dexamethasone or by long-term culture in serum-containing medium. Stimulation of the cAMP-dependent signalling allowed us to isolate clonal pancreatic ductal cells from adult mice. These cells are able to partially differentiate into endocrine cells, exocrine cells and hepatocyte-like cells and are therefore considered to have the characteristics of endodermal progenitor cells.
Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

PubMed

Lübberstedt, Marc; Müller-Vieira, Ursula; Biemel, Klaus M; Darnell, Malin; Hoffmann, Stefan A; Knöspel, Fanny; Wönne, Eva C; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B; Zeilinger, Katrin

2015-09-01

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions. Copyright © 2012 John Wiley & Sons, Ltd.
Extracellular small RNAs: what, where, why?

PubMed Central

Hoy, Anna M.; Buck, Amy H.

2012-01-01

miRNAs (microRNAs) are a class of small RNA that regulate gene expression by binding to mRNAs and modulating the precise amount of proteins that get expressed in a cell at a given time. This form of gene regulation plays an important role in developmental systems and is critical for the proper function of numerous biological pathways. Although miRNAs exert their functions inside the cell, these and other classes of RNA are found in body fluids in a cell-free form that is resistant to degradation by RNases. A broad range of cell types have also been shown to secrete miRNAs in association with components of the RISC (RNA-induced silencing complex) and/or encapsulation within vesicles, which can be taken up by other cells. In the present paper, we provide an overview of the properties of extracellular miRNAs in relation to their capacity as biomarkers, stability against degradation and mediators of cell–cell communication. PMID:22817753
Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

PubMed

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.
Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells

PubMed Central

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

2016-01-01

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible. PMID:26942051
High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format

PubMed Central

Quast, Robert B.; Sonnabend, Andrei; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

2016-01-01

Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2. PMID:27456041
Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.
Structural and functional characterisation of FOXO/Acan-DAF-16 from the parasitic nematode Angiostrongylus cantonensis.

PubMed

Yan, Baolong; Sun, Weiwei; Yan, Lanzhu; Zhang, Liangliang; Zheng, Yuan; Zeng, Yuzhen; Huang, Huicong; Liang, Shaohui

2016-12-01

Fork head box transcription factors subfamily O (FoxO) is regarded to be significant in cell-cycle control, cell differentiation, ageing, stress response, apoptosis, tumour formation and DNA damage repair. In the free-living nematode Caenorhabditis elegans, the FoxO transcription factor is encoded by Ce-daf-16, which is negatively regulated by insulin-like signaling (IIS) and involved in promoting dauer formation through bringing about its hundreds of downstream genes expression. In nematode parasites, orthologues of daf-16 from several species have been identified, with functions in rescue of dauer phenotypes determined in a surrogate system C. elegans. In this study, we identified the FoxO encoding gene, Acan-daf-16, from the parasitic nematode Angiostrongylus cantonensis, and determined the genomic structures, transcripts and functions far more thorough in longevity, stress resistance and dauer formation. Acan-daf-16 encodes two proteins, Acan-DAF-16A and Acan-DAF-16B, consisting of 555 and 491 amino acids, respectively. Both isoforms possess the highly conserved fork head domains. Acan-daf-16A and Acan-daf-16B are expressed from distinct promoters. The expression patterns of Acan-daf-16 isoforms in the C. elegans surrogate system showed that p Acan-daf-16a:gfp was expressed in all cells of C. elegans, including the pharynx, and the expression of p Acan-daf-16b:gfp was restricted to the pharynx. In addition to the same genomic organization to the orthologue in C. elegans, Ce-daf-16, both Acan-DAF-16 isoforms could restore the C. elegans daf-16(mg54) mutation in longevity, dauer formation and stress resistance, in spite of the partial complementation of Acan-DAF-16B isoform in longevity. These findings provide further evidence of the functional conservation of DAF-16s between parasitic nematodes and the free-living nematode C. elegans. Copyright © 2016. Published by Elsevier B.V.
Blockade of LAG3 enhances responses of tumor-infiltrating T cells in mismatch repair-proficient liver metastases of colorectal cancer

PubMed Central

Noordam, Lisanne; Sprengers, Dave; Boor, Patrick P. C.; Mancham, Shanta; Menon, Anand G.; Lange, Johan F.; Burger, Pim J. W. A.; Brandt, Alexandra; Galjart, Boris; Kwekkeboom, Jaap; Bruno, Marco J.

2018-01-01

ABSTRACT Purpose: Liver metastasis develops in >50% of patients with colorectal cancer (CRC), and is a leading cause of CRC-related mortality. We aimed to identify which inhibitory immune checkpoint pathways can be targeted to enhance functionality of intra-tumoral T-cells in mismatch repair-proficient liver metastases of colorectal cancer (LM-CRC). Methodology: Intra-tumoral expression of multiple inhibitory molecules was compared among mismatch repair-proficient LM-CRC, peritoneal metastases of colorectal cancer (PM-CRC) and primary CRC. Expression of inhibitory molecules was also analyzed on leukocytes isolated from paired resected metastatic liver tumors, tumor-free liver tissues, and blood of patients with mismatch repair-proficient LM-CRC. The effects of blocking inhibitory pathways on tumor-infiltrating T-cell responses were studied in ex vivo functional assays. Results: Mismatch repair-proficient LM-CRC showed higher expression of inhibitory receptors on intra-tumoral T-cells and contained higher proportions of CD8+ T-cells, dendritic cells and monocytes than mismatch repair-proficient primary CRC and/or PM-CRC. Inhibitory receptors LAG3, PD-1, TIM3 and CTLA4 were higher expressed on CD8+ T-cells, CD4+ T-helper and/or regulatory T-cells in LM-CRC tumors compared with tumor-free liver and blood. Antibody blockade of LAG3 or PD-L1 increased proliferation and effector cytokine production of intra-tumoral T-cells isolated from LM-CRC in response to both polyclonal and autologous tumor-specific stimulations. Higher LAG3 expression on intra-tumoral CD8+ T-cells associated with longer progression-free survival of LM-CRC patients. Conclusion: Mismatch repair-proficient LM-CRC may be more sensitive to immune checkpoint inhibitors than mismatch repair-proficient primary CRC. Blocking LAG3 enhances tumor-infiltrating T-cell responses of mismatch repair-proficient LM-CRC, and therefore may be a new promising immunotherapeutic target for LM-CRC.
Targeting of free fatty acid receptor 1 in EOC: A novel strategy to restrict the adipocyte-EOC dependence.

PubMed

Munkarah, Adnan; Mert, Ismail; Chhina, Jasdeep; Hamid, Suhail; Poisson, Laila; Hensley-Alford, Sharon; Giri, Shailendra; Rattan, Ramandeep

2016-04-01

Adipocyte derived free fatty acids (FFA) promote epithelial ovarian cancer (EOC) by acting as a fuel source to support the energy requirement of the cancer cells. FFA may also exert biological effects through signaling pathways. Recently, a family of FFA activated G-protein coupled receptors (FFAR/GPCRs) was identified. Our objective was to investigate the role of FFAR/GPCRs in EOC and assess their potential as therapeutic targets. The mRNA (RT-PCR) expression of FFAR/GPCR family members (FFAR1/GPR40; FFAR2/GPR43, FFAR3/GPR41, FFAR4/GPR120 and GPR84) was examined in: (1) a syngeneic mouse model of EOC fed high energy diet (60% fat) or regular diet (30% fat), (2) EOC cell lines exposed to free fatty acids and (3) specimens from 13 histologically normal ovaries and 28 high grade ovarian serous carcinomas. The GPR 40 antagonist, GW1100, was used to inhibit FFAR1/GPR40 and cell survival was assayed by MTT in various cell lines. High Grade Serous carcinoma specimens expressed significantly increased GPR40 compared to normal ovaries (p=0.0020). Higher expression was noted in advanced stage disease. ID8 ovarian tumors from mice fed with high fat diet also showed higher GPR40 expression. Exposing EOC cells to FFAs, increased GPR40 expression. Treatment of EOC cell lines with GW100 resulted in growth inhibition and was associated with an alteration in their energy metabolism. FFA-induced cancer cell growth may be partly mediated through FFAR1/GPR40. Targeting of FFAR1/GPR40 may be an attractive treatment strategy in EOC, and possibly offers a targeted treatment for a subset of EOC patients. Copyright © 2016 Elsevier Inc. All rights reserved.
Glutathione peroxidase 4 overexpression inhibits ROS-induced cell death in diffuse large B-cell lymphoma.

PubMed

Kinowaki, Yuko; Kurata, Morito; Ishibashi, Sachiko; Ikeda, Masumi; Tatsuzawa, Anna; Yamamoto, Masahide; Miura, Osamu; Kitagawa, Masanobu; Yamamoto, Kouhei

2018-02-20

Regulation of oxidative stress and redox systems has important roles in carcinogenesis and cancer progression, and for this reason has attracted much attention as a new area of cancer therapeutic targets. Glutathione peroxidase 4 (GPX4), an antioxidant enzyme, has biological important functions such as signaling cell death by suppressing peroxidation of membrane phospholipids. However, few studies exist on the expression and clinical relevance of GPX4 in malignant lymphomas such as diffuse large B-cell lymphoma. In this study, we assessed the expression of GPX4 immunohistochemically. GPX4 was expressed in 35.5% (33/93) cases of diffuse large B-cell lymphoma. The GPX4-positive group had poor overall survival (P = 0.0032) and progression-free survival (P = 0.0004) compared with those of the GPX4-negative group. In a combined analysis of GPX4 and 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, there was a negative correlation between GPX4 and 8-hydroxydeoxyguanosine (P = 0.0009). The GPX4-positive and 8-hydroxydeoxyguanosine-negative groups had a significantly worse prognosis than the other groups in both overall survival (P = 0.0170) and progression-free survival (P = 0.0005). These results suggest that the overexpression of GPX4 is an independent prognostic predictor in diffuse large B-cell lymphoma. Furthermore, in vitro analysis demonstrated that GPX4-overexpressing cells were resistant to reactive oxygen species-induced cell death (P = 0.0360). Conversely, GPX4-knockdown cells were sensitive to reactive oxygen species-induced cell death (P = 0.0111). From these data, we conclude that GPX4 regulates reactive oxygen species-induced cell death. Our results suggest a novel therapeutic strategy using the mechanism of ferroptosis, as well as a novel prognostic predictor of diffuse large B-cell lymphoma.
Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043
Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells

NASA Astrophysics Data System (ADS)

Yu, Xinwei; Feizpour, Amin; Ramirez, Nora-Guadalupe P.; Wu, Linxi; Akiyama, Hisashi; Xu, Fangda; Gummuluru, Suryaram; Reinhard, Björn M.

2014-06-01

Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.
Gene expression profiling analysis of the effects of low-intensity pulsed ultrasound on induced pluripotent stem cell-derived neural crest stem cells.

PubMed

Xia, Bin; Zou, Yang; Xu, Zhiling; Lv, Yonggang

2017-11-01

Low-intensity pulsed ultrasound (LIPUS) is a noninvasive technique that has been shown to affect cell proliferation, migration, and differentiation and promote the regeneration of damaged peripheral nerve. Our previous studies had proved that LIPUS can significantly promote the neural differentiation of induced pluripotent stem cell-derived neural crest stem cells (iPSCs-NCSCs) and enhance the repair of rat-transected sciatic nerve. To further explore the underlying mechanisms of LIPUS treatment of iPSCs-NCSCs, this study reported the gene expression profiling analysis of iPSCs-NCSCs before and after LIPUS treatment using the RNA-sequencing (RNA-Seq) method. It was found that expression of 76 genes of iPSCs-NCSCs cultured in a serum-free neural induction medium and expression of 21 genes of iPSCs-NCSCs cultured in a neuronal differentiation medium were significantly changed by LIPUS treatment. The differentially expressed genes are related to angiogenesis, nervous system activity and functions, cell activities, and so on. The RNA-seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). High correlation was observed between the results obtained from qRT-PCR and RNA-Seq. This study presented new information on the global gene expression patterns of iPSCs-NCSCs after LIPUS treatment and may expand the understanding of the complex molecular mechanism of LIPUS treatment of iPSCs-NCSCs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.
Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines.

PubMed

Tarjan, Gabor; Haines, G Kenneth; Vesper, Benjamin J; Xue, Jiaping; Altman, Michael B; Yarmolyuk, Yaroslav R; Khurram, Huma; Elseth, Kim M; Roeske, John C; Aydogan, Bulent; Radosevich, James A

2011-02-01

It is not understood why some head and neck squamous cell carcinomas, despite having identical morphology, demonstrate different tumor aggressiveness, including radioresistance. High levels of the free radical nitric oxide (NO) and increased expression of the NO-producing enzyme nitric oxide synthase (NOS) have been implicated in tumor progression. We previously adapted three human tongue cancer cell lines to high NO (HNO) levels by gradually exposing them to increasing concentrations of an NO donor; the HNO cells grew faster than their corresponding untreated ("parent") cells, despite being morphologically identical. Herein we initially characterize the HNO cells and compare the biological properties of the HNO and parent cells. HNO/parent cell line pairs were analyzed for cell cycle distribution, DNA damage, X-ray and ultraviolet radiation response, and expression of key cellular enzymes, including NOS, p53, glutathione S-transferase-pi (GST-pi), apurinic/apyrimidinic endonuclease-1 (APE1), and checkpoint kinases (Chk1, Chk2). While some of these properties were cell line-specific, the HNO cells typically exhibited properties associated with a more aggressive behavior profile than the parent cells (greater S-phase percentage, radioresistance, and elevated expression of GST-pi/APE1/Chk1/Chk2). To correlate these findings with conditions in primary tumors, we examined the NOS, GST-pi, and APE1 expression in human tongue squamous cell carcinomas. A majority of the clinical samples exhibited elevated expression levels of these enzymes. Together, the results herein suggest cancer cells exposed to HNO levels can develop resistance to free radicals by upregulating protective mechanisms, such as GST-pi and APE1. These upregulated defense mechanisms may contribute to their aggressive expression profile.
Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

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

Deng, Xuefeng, E-mail: dengxfdoctor@hotmail.com; Department of Cardio-thoracic Surgery, Affiliated Hospital of Academy of Military Medical Sciences; Ma, Qunfeng

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level ofmore » MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression. - Highlights: • MSF expression was upregulated in NSCLC and correlated with TNM stages. • MSF may be a new biomarker for NSCLC progression. • MSF promoted migration and invasion in A549 cells, independent of MMP-2/MMP-9 expression.« less
Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

PubMed Central

Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2012-01-01

The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500–2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation. PMID:22802633
Single cell digital polymerase chain reaction on self-priming compartmentalization chip

PubMed Central

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%–4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease. PMID:28191267
Single cell digital polymerase chain reaction on self-priming compartmentalization chip.

PubMed

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%-4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease.
Expression and function of survivin in canine osteosarcoma.

PubMed

Shoeneman, Jenette K; Ehrhart, E J; Eickhoff, Jens C; Charles, J B; Powers, Barbara E; Thamm, Douglas H

2012-01-01

Osteosarcoma has a high mortality rate and remains in need of more effective therapeutic approaches. Survivin is an inhibitor of apoptosis family member protein that blocks apoptosis and drives proliferation in human cancer cells where it is commonly elevated. In this study, we illustrate the superiority of a canine osteosarcoma model as a translational tool for evaluating survivin-directed therapies, owing to the striking similarities in gross and microscopic appearance, biologic behavior, gene expression, and signaling pathway alterations. Elevated survivin expression in primary canine osteosarcoma tissue correlated with increased histologic grade and mitotic index and a decreased disease-free interval (DFI). Survivin attenuation in canine osteosarcoma cells inhibited cell-cycle progression, increased apoptosis, mitotic arrest, and chemosensitivity, and cooperated with chemotherapy to significantly improve in vivo tumor control. Our findings illustrate the utility of a canine system to more accurately model human osteosarcoma and strongly suggest that survivin-directed therapies might be highly effective in its treatment. ©2011 AACR.

Morphological diversity of the avian foot is related with the pattern of msx gene expression in the developing autopod.

PubMed

Gañan, Y; Macias, D; Basco, R D; Merino, R; Hurle, J M

1998-04-01

The formation of the digits in amniota embryos is accompanied by apoptotic cell death of the interdigital mesoderm triggered through BMP signaling. Differences in the intensity of this apoptotic process account for the establishment of the different morphological types of feet observed in amniota (i.e., free-digits, webbed digits, lobulated digits). The molecular basis accounting for the differential pattern of interdigital cell death remains uncertain since the reduction of cell death in species with webbed digits is not accompanied by a parallel reduction in the pattern of expression of bmp genes in the interdigital regions. In this study we show that the duck interdigital web mesoderm exhibits an attenuated response to both BMP-induced apoptosis and TGFbeta-induced chondrogenesis in comparison with species with free digits. The attenuated response to these signals is accompanied by a reduced pattern of expression of msx-1 and msx-2 genes. Local application of FGF in the duck interdigit expands the domain of msx-2 expression but not the domain of msx-1 expression. This change in the expression of msx-2 is followed by a parallel increase in spontaneous and exogenous BMP-induced interdigital cell death, while the chondrogenic response to TGFbetas is unchanged. The regression of AER, as deduced by the pattern of extinction of fgf-8 expression, takes place in a similar fashion in the chick and duck regardless of the differences in interdigital cell death and msx gene expression. Implantation of BMP-beads in the distal limb mesoderm induces AER regression in both the chick and duck. This finding suggests an additional role for BMPs in the physiological regression of the AER. It is proposed that the formation of webbed vs free-digit feet in amniota results from a premature differentiation of the interdigital mesoderm into connective tissue caused by a reduced expression of msx genes in the developing autopod. Copyright 1998 Academic Press.
Normalizing the environment recapitulates adult human immune traits in laboratory mice.

PubMed

Beura, Lalit K; Hamilton, Sara E; Bi, Kevin; Schenkel, Jason M; Odumade, Oludare A; Casey, Kerry A; Thompson, Emily A; Fraser, Kathryn A; Rosato, Pamela C; Filali-Mouhim, Ali; Sekaly, Rafick P; Jenkins, Marc K; Vezys, Vaiva; Haining, W Nicholas; Jameson, Stephen C; Masopust, David

2016-04-28

Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside. Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice--like newborn, but not adult, humans--lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.
Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

PubMed

Lee, Waisin; Xu, Mingjing; Li, Yue; Gu, Yong; Chen, Jianping; Wong, Derek; Fung, Peter C W; Shen, Jiangang

2011-10-01

Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death. Copyright © 2011 Elsevier Inc. All rights reserved.
Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.

PubMed

Phondeechareon, Tanapol; Wattanapanitch, Methichit; U-Pratya, Yaowalak; Damkham, Chanapa; Klincumhom, Nuttha; Lorthongpanich, Chanchao; Kheolamai, Pakpoom; Laowtammathron, Chuti; Issaragrisil, Surapol

2016-10-01

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH, however, lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore, other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs), characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming, and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation, the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients.
Rational design of engineered microbial cell surface multi-enzyme co-display system for sustainable NADH regeneration from low-cost biomass.

PubMed

Han, Lei; Liang, Bo; Song, Jianxia

2018-02-01

As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16 g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.
Activation of D4 dopamine receptor decreases AT1 angiotensin II receptor expression in rat renal proximal tubule cells

PubMed Central

Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D.; Jose, Pedro A.; Zeng, Chunyu

2014-01-01

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal AT1 receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubules (RPTs) cells from Wistar-Kyoto (WKY) rats but the D4 receptor regulation of AT1 receptor is aberrant in RPT cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na+-K+ ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na+-K+ ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na+-K+ ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na+-K+ ATPase activity in SHR cells. This was confirmed in vivo; pre-treatment with PD128077 for one week augmented the anti-hypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension. PMID:25368031
The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

PubMed

Kim, Jong-Hyun; Lee, Sang-Hee; Sohn, Hae-Jin; Lee, Jinyoung; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

2012-12-01

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.
Analysis of the expression and antioxidant activity of 2-Cys peroxiredoxin protein in Fasciola gigantica.

PubMed

Sangpairoj, Kant; Changklungmoa, Narin; Vanichviriyakit, Rapeepun; Sobhon, Prasert; Chaithirayanon, Kulathida

2014-05-01

2-Cys peroxiredoxin (Prx) is the main antioxidant enzyme in Fasciola species for detoxifying hydrogen peroxide which is generated from the hosts' immune effector cells and the parasites' own metabolism. In this study, the recombinant Prx protein from Fasciola gigantica (rFgPrx-2) was expressed and purified in a prokaryotic expression system. This recombinant protein with molecular weight of 26 kDa was enzymatically active in reduction of hydrogen peroxide both in presence of thioredoxin and glutathione systems, and also protected the supercoiled plasmid DNA from oxidative damage in metal-catalyzed oxidation (MCO) system in a concentration-dependent manner. By immunoblotting, using antibody against rFgPrx-2 as probe, a native FgPrxs, whose MW at 25 kDa, was detected in all developmental stages of the parasite. Concentrations of native FgPrxs were increasing in all stages reaching highest level in adult stage. The antibody also showed cross reactivities with corresponding proteins in some cattle helminthes. Natural antibody to FgPrxs could be detected in the sera of mice at 3 and 4 weeks after infection with F. gigantica metacercariae. By immunofluorescence, FgPrxs was highly expressed in tegument and tegumental cells, parenchyma, moderately expressed in cecal epithelial cells in early, juvenile and adult worms. Furthermore, FgPrxs was also detected in the female reproductive organs, including eggs, ovary, vitelline cells, and testis, suggesting that FgPrxs might play an essential role in protecting parasite's tissues from free radical attack during their life cycle. Thus, FgPrxs is one potential candidate for drug therapy and vaccine development. Copyright © 2014 Elsevier Inc. All rights reserved.
Hydrogen production by recombinant Escherichia coli strains

PubMed Central

Maeda, Toshinari; Sanchez‐Torres, Viviana; Wood, Thomas K.

2012-01-01

Summary The production of hydrogen via microbial biotechnology is an active field of research. Given its ease of manipulation, the best‐studied bacterium Escherichia coli has become a workhorse for enhanced hydrogen production through metabolic engineering, heterologous gene expression, adaptive evolution, and protein engineering. Herein, the utility of E. coli strains to produce hydrogen, via native hydrogenases or heterologous ones, is reviewed. In addition, potential strategies for increasing hydrogen production are outlined and whole‐cell systems and cell‐free systems are compared. PMID:21895995
Proteinaceous and oligosaccharidic elicitors induce different calcium signatures in the nucleus of tobacco cells.

PubMed

Lecourieux, David; Lamotte, Olivier; Bourque, Stéphane; Wendehenne, David; Mazars, Christian; Ranjeva, Raoul; Pugin, Alain

2005-12-01

We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627-2641]. These data suggested that in response to elicitors, Ca2+, as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca2+](nuc)) in response to elicitors. Two types of elicitors are compared: proteins leading to necrosis including four elicitins and harpin, and non-necrotic elicitors including flagellin (flg22) and two oligosaccharidic elicitors, namely the oligogalacturonides (OGs) and the beta-1,3-glucan laminarin. Our data indicate that the proteinaceous elicitors induced a pronounced and sustainable [Ca2+](nuc) elevation, relative to the small effects of oligosaccharidic elicitors. This [Ca2+](nuc) elevation, which seems insufficient to induce cell death, is unlikely to result directly from the diffusion of calcium from the cytosol. The [Ca2+](nuc) rise depends on free cytosolic calcium, IP3, and active oxygen species (AOS) but is independent of nitric oxide.
Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

NASA Astrophysics Data System (ADS)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.
Cell-Free and In Vivo Characterization of Lux, Las, and Rpa Quorum Activation Systems in E. coli.

PubMed

Halleran, Andrew D; Murray, Richard M

2018-02-16

Synthetic biologists have turned toward quorum systems as a path for building sophisticated microbial consortia that exhibit group decision making. Currently, however, even the most complex consortium circuits rely on only one or two quorum sensing systems, greatly restricting the available design space. High-throughput characterization of available quorum sensing systems is useful for finding compatible sets of systems that are suitable for a defined circuit architecture. Recently, cell-free systems have gained popularity as a test-bed for rapid prototyping of genetic circuitry. We take advantage of the transcription-translation cell-free system to characterize three commonly used Lux-type quorum activators, Lux, Las, and Rpa. We then compare the cell-free characterization to results obtained in vivo. We find significant genetic crosstalk in both the Las and Rpa systems and substantial signal crosstalk in Lux activation. We show that cell-free characterization predicts crosstalk observed in vivo.
Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

PubMed

Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

2014-02-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. Copyright © 2013 Elsevier Ltd. All rights reserved.
In situ synthesis of protein arrays.

PubMed

He, Mingyue; Stoevesandt, Oda; Taussig, Michael J

2008-02-01

In situ or on-chip protein array methods use cell free expression systems to produce proteins directly onto an immobilising surface from co-distributed or pre-arrayed DNA or RNA, enabling protein arrays to be created on demand. These methods address three issues in protein array technology: (i) efficient protein expression and availability, (ii) functional protein immobilisation and purification in a single step and (iii) protein on-chip stability over time. By simultaneously expressing and immobilising many proteins in parallel on the chip surface, the laborious and often costly processes of DNA cloning, expression and separate protein purification are avoided. Recently employed methods reviewed are PISA (protein in situ array) and NAPPA (nucleic acid programmable protein array) from DNA and puromycin-mediated immobilisation from mRNA.
Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

NASA Astrophysics Data System (ADS)

Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

2014-01-01

We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.
The free-energy cost of interaction between DNA loops.

PubMed

Huang, Lifang; Liu, Peijiang; Yuan, Zhanjiang; Zhou, Tianshou; Yu, Jianshe

2017-10-03

From the viewpoint of thermodynamics, the formation of DNA loops and the interaction between them, which are all non-equilibrium processes, result in the change of free energy, affecting gene expression and further cell-to-cell variability as observed experimentally. However, how these processes dissipate free energy remains largely unclear. Here, by analyzing a mechanic model that maps three fundamental topologies of two interacting DNA loops into a 4-state model of gene transcription, we first show that a longer DNA loop needs more mean free energy consumption. Then, independent of the type of interacting two DNA loops (nested, side-by-side or alternating), the promotion between them always consumes less mean free energy whereas the suppression dissipates more mean free energy. More interestingly, we find that in contrast to the mechanism of direct looping between promoter and enhancer, the facilitated-tracking mechanism dissipates less mean free energy but enhances the mean mRNA expression, justifying the facilitated-tracking hypothesis, a long-standing debate in biology. Based on minimal energy principle, we thus speculate that organisms would utilize the mechanisms of loop-loop promotion and facilitated tracking to survive in complex environments. Our studies provide insights into the understanding of gene expression regulation mechanism from the view of energy consumption.
Overexpression of caldesmon is associated with tumor progression in patients with primary non-muscle-invasive bladder cancer

PubMed Central

Lee, Myung-Shin; Lee, Jisu; Kim, Joo Heon; Kim, Won Tae; Kim, Wun-Jae; Ahn, Hanjong; Park, Jinsung

2015-01-01

The expression and function of caldesmon (CAD) in urothelial bladder carcinoma (BC) have not been reported. Here, we investigated the expression, prognostic value, and potential functional mechanism of CAD in primary non-muscle-invasive bladder cancer (NMIBC). Protein profiling of tissue samples using antibody microarrays showed significantly higher CAD expression in muscle-invasive BC tissues compared with NMIBC tissues. We then validated the CAD expression in BC cells by immunohistochemistry analysis using paraffin-embedded tissue blocks and western blots using BC cell lines. In addition, we examined the expression of CAD variants by reverse transcription-polymerase chain reaction, and confirmed the expression of low-molecular-weight isoforms (L-CAD), specifically encoded by WI-38 L-CAD II (transcript variant 2), in BC cells. Survival analysis in an independent primary NMIBC cohort comprising 132 patients showed that positive CAD expression was significantly associated with poorer prognosis than no CAD expression with regard to recurrence- and progression-free survival (p = 0.001 and 0.014, respectively). Multivariate analyses further indicated that positive CAD expression was an independent predictor of progression-free survival (p = 0.032; HR = 5.983). Data obtained from in vitro silencing and overexpression studies indicated that L-CAD promotes migration and invasiveness of BC cells. Immunofluorescence assays showed dramatic structural changes in the actin cytoskeleton of BC cells after L-CAD overexpression. Our findings collectively suggest that L-CAD overexpression in primary NMIBC is significantly associated with tumor progression and that a possible mechanism for L-CAD's activity is implicated in increased cell motility and invasive characteristics through morphological changes in BC cells. PMID:26430961
Altered expression of epithelial mesenchymal transition and pluripotent associated markers by sex steroid hormones in human embryonic stem cells.

PubMed

Jeon, So-Ye; Hwang, Kyung-A; Kim, Cho-Won; Jeung, Eui-Bae; Choi, Kyung-Chul

2017-07-01

Embryonic stem (ES) cells are pluripotent stem cells derived from a developmental stage of pre‑implanted embryos. The present study investigated the effect of female sex steroid hormones on the characteristics of human ES cells by using a feeder‑free culture protocol. In a feeder‑free condition without sex hormones, human ES cells assumed the form of tightly packed cells that grow in a monolayer. The cells had clean and defined edges with no evidence of differentiation and expressed several markers specific for undifferentiated ES cells including POU class 5 homeobox 1 (POU5F1), sex determining region Y‑box 2 (SOX2) and NANOG homeobox (NANOG). It was then investigated if female sex steroid hormones including 17β‑estradiol (E2) and progesterone (P4) altered the protein expression of epithelial-mesenchymal transition (EMT) associated markers in addition to pluripotency markers including POU5F1, SOX2 and NANOG in human ES cells. The protein expression levels of N‑cadherin, Snail and Slug were increased while E‑cadherin expression was decreased by treatment of E2 or P4, and the expression levels of POU5F1, SOX2 and NANOG were decreased by the treatment of E2 or P4. When E2 and P4 were treated in combination with an estrogen receptor inhibitor (ICI 182,780) and progesterone receptor inhibitor (RU486) respectively, their effects on EMT and pluripotency of ES cells were restored to control levels. The results suggested that E2 and P4 may regulate EMT and pluripotency of human ES cells by mediating their receptors. The present study may aid in the understanding of the role of sex steroid hormones in the cellular biology of human ES cells.
Light-patterning of synthetic tissues with single droplet resolution.

PubMed

Booth, Michael J; Restrepo Schild, Vanessa; Box, Stuart J; Bayley, Hagan

2017-08-24

Synthetic tissues can be generated by forming networks of aqueous droplets in lipid-containing oil. Each droplet contains a cell-free expression system and is connected to its neighbor through a lipid bilayer. In the present work, we have demonstrated precise external control of such networks by activating protein expression within single droplets, by using light-activated DNA to encode either a fluorescent or a pore-forming protein. By controlling the extent of activation, synthetic tissues were generated with graded levels of protein expression in patterns of single droplets. Further, we have demonstrated reversible activation within individual compartments in synthetic tissues by turning a fluorescent protein on-and-off. This is the first example of the high-resolution patterning of droplet networks, following their formation. Single-droplet control will be essential to power subsets of compartments within synthetic tissues or to stimulate subsets of cells when synthetic tissues are interfaced with living tissues.
The Staphylococcus aureus Response to Unsaturated Long Chain Free Fatty Acids: Survival Mechanisms and Virulence Implications

PubMed Central

Kenny, John G.; Ward, Deborah; Josefsson, Elisabet; Jonsson, Ing-Marie; Hinds, Jason; Rees, Huw H.; Lindsay, Jodi A.; Tarkowski, Andrej; Horsburgh, Malcolm J.

2009-01-01

Staphylococcus aureus is an important human commensal and opportunistic pathogen responsible for a wide range of infections. Long chain unsaturated free fatty acids represent a barrier to colonisation and infection by S. aureus and act as an antimicrobial component of the innate immune system where they are found on epithelial surfaces and in abscesses. Despite many contradictory reports, the precise anti-staphylococcal mode of action of free fatty acids remains undetermined. In this study, transcriptional (microarrays and qRT-PCR) and translational (proteomics) analyses were applied to ascertain the response of S. aureus to a range of free fatty acids. An increase in expression of the σB and CtsR stress response regulons was observed. This included increased expression of genes associated with staphyloxanthin synthesis, which has been linked to membrane stabilisation. Similarly, up-regulation of genes involved in capsule formation was recorded as were significant changes in the expression of genes associated with peptidoglycan synthesis and regulation. Overall, alterations were recorded predominantly in pathways involved in cellular energetics. In addition, sensitivity to linoleic acid of a range of defined (sigB, arcA, sasF, sarA, agr, crtM) and transposon-derived mutants (vraE, SAR2632) was determined. Taken together, these data indicate a common mode of action for long chain unsaturated fatty acids that involves disruption of the cell membrane, leading to interference with energy production within the bacterial cell. Contrary to data reported for other strains, the clinically important EMRSA-16 strain MRSA252 used in this study showed an increase in expression of the important virulence regulator RNAIII following all of the treatment conditions tested. An adaptive response by S. aureus of reducing cell surface hydrophobicity was also observed. Two fatty acid sensitive mutants created during this study were also shown to diplay altered pathogenesis as assessed by a murine arthritis model. Differences in the prevalence and clinical importance of S. aureus strains might partly be explained by their responses to antimicrobial fatty acids. PMID:19183815

The histone deacetylase inhibiting drug Entinostat induces lipid accumulation in differentiated HepaRG cells

NASA Astrophysics Data System (ADS)

Nunn, Abigail D. G.; Scopigno, Tullio; Pediconi, Natalia; Levrero, Massimo; Hagman, Henning; Kiskis, Juris; Enejder, Annika

2016-06-01

Dietary overload of toxic, free metabolic intermediates leads to disrupted insulin signalling and fatty liver disease. However, it was recently reported that this pathway might not be universal: depletion of histone deacetylase (HDAC) enhances insulin sensitivity alongside hepatic lipid accumulation in mice, but the mechanistic role of microscopic lipid structure in this effect remains unclear. Here we study the effect of Entinostat, a synthetic HDAC inhibitor undergoing clinical trials, on hepatic lipid metabolism in the paradigmatic HepaRG liver cell line. Specifically, we statistically quantify lipid droplet morphology at single cell level utilizing label-free microscopy, coherent anti-Stokes Raman scattering, supported by gene expression. We observe Entinostat efficiently rerouting carbohydrates and free-fatty acids into lipid droplets, upregulating lipid coat protein gene Plin4, and relocating droplets nearer to the nucleus. Our results demonstrate the power of Entinostat to promote lipid synthesis and storage, allowing reduced systemic sugar levels and sequestration of toxic metabolites within protected protein-coated droplets, suggesting a potential therapeutic strategy for diseases such as diabetes and metabolic syndrome.
Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts

PubMed Central

Ahn, Jin-Ho; Hwang, Mi-Yeon; Lee, Kyung-Ho; Choi, Cha-Yong; Kim, Dong-Myung

2007-01-01

This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours. PMID:17185295
Increased signaling entropy in cancer requires the scale-free property of protein interaction networks.

PubMed

Teschendorff, Andrew E; Banerji, Christopher R S; Severini, Simone; Kuehn, Reimer; Sollich, Peter

2015-04-28

One of the key characteristics of cancer cells is an increased phenotypic plasticity, driven by underlying genetic and epigenetic perturbations. However, at a systems-level it is unclear how these perturbations give rise to the observed increased plasticity. Elucidating such systems-level principles is key for an improved understanding of cancer. Recently, it has been shown that signaling entropy, an overall measure of signaling pathway promiscuity, and computable from integrating a sample's gene expression profile with a protein interaction network, correlates with phenotypic plasticity and is increased in cancer compared to normal tissue. Here we develop a computational framework for studying the effects of network perturbations on signaling entropy. We demonstrate that the increased signaling entropy of cancer is driven by two factors: (i) the scale-free (or near scale-free) topology of the interaction network, and (ii) a subtle positive correlation between differential gene expression and node connectivity. Indeed, we show that if protein interaction networks were random graphs, described by Poisson degree distributions, that cancer would generally not exhibit an increased signaling entropy. In summary, this work exposes a deep connection between cancer, signaling entropy and interaction network topology.
Increased signaling entropy in cancer requires the scale-free property of protein interaction networks

PubMed Central

Teschendorff, Andrew E.; Banerji, Christopher R. S.; Severini, Simone; Kuehn, Reimer; Sollich, Peter

2015-01-01

One of the key characteristics of cancer cells is an increased phenotypic plasticity, driven by underlying genetic and epigenetic perturbations. However, at a systems-level it is unclear how these perturbations give rise to the observed increased plasticity. Elucidating such systems-level principles is key for an improved understanding of cancer. Recently, it has been shown that signaling entropy, an overall measure of signaling pathway promiscuity, and computable from integrating a sample's gene expression profile with a protein interaction network, correlates with phenotypic plasticity and is increased in cancer compared to normal tissue. Here we develop a computational framework for studying the effects of network perturbations on signaling entropy. We demonstrate that the increased signaling entropy of cancer is driven by two factors: (i) the scale-free (or near scale-free) topology of the interaction network, and (ii) a subtle positive correlation between differential gene expression and node connectivity. Indeed, we show that if protein interaction networks were random graphs, described by Poisson degree distributions, that cancer would generally not exhibit an increased signaling entropy. In summary, this work exposes a deep connection between cancer, signaling entropy and interaction network topology. PMID:25919796
Endocannabinoids: Multi-scaled, Global Homeostatic Regulators of Cells and Society

NASA Astrophysics Data System (ADS)

Melamede, Robert

Living systems are far from equilibrium open systems that exhibit many scales of emergent behavior. They may be abstractly viewed as a complex weave of dissipative structures that maintain organization by passing electrons from reduced hydrocarbons to oxygen. Free radicals are unavoidable byproducts of biological electron flow. Due to their highly reactive chemical properties, free radicals modify all classes of biological molecules (carbohydrates, lipids, nucleic acids, and proteins). As a result, free radicals are destructive. The generally disruptive nature of free radicals makes them the "friction of life." As such, they are believed to be the etiological agents behind age related illnesses such as cardiovascular, immunological, and neurological diseases, cancer, and ageing itself. Free radicals also play a critical constructive role in living systems. From a thermodynamic perspective, life can only exist if a living system takes in sufficient negative entropy from its environment to overcome the obligatory increase in entropy that would result if the system could not appropriately exchange mass, energy and information with its environment. Free radicals are generated in response to perturbations in the relationship between a living system and its environment. However, evolution has selected for biological response systems to free radicals so that the cellular biochemistry can adapt to environmental perturbations by modifying cellular gene expression and biochemistry. Endocannabinoids are marijuana-like compounds that have their origins hundreds of millions of years in the evolutionary past. They serve as fundamental modulators of energy homeostasis in all vertebrates. Their widespread biological activities may often be attributed to their ability to minimize the negative consequences of free radicals.
Entrapment of an EGFR inhibitor into nanostructured lipid carriers (NLC) improves its antitumor activity against human hepatocarcinoma cells

PubMed Central

2014-01-01

Background In hepatocellular carcinoma (HCC), different signaling pathways are de-regulated, and among them, the expression of the epidermal growth factor receptor (EGFR). Tyrphostin AG-1478 is a lipophilic low molecular weight inhibitor of EGFR, preferentially acting on liver tumor cells. In order to overcome its poor drug solubility and thus improving its anticancer activity, it was entrapped into nanostructured lipid carriers (NLC) by using safe ingredients for parenteral delivery. Results Nanostructured lipid carriers (NLC) carrying tyrphostin AG-1478 were prepared by using the nanoprecipitation method and different matrix compositions. The best system in terms of mean size, PDI, zeta potential, drug loading and release profile was chosen to evaluate the anti-proliferative effect of drug-loaded NLC versus free drug on human hepatocellular carcinoma HA22T/VGH cells. Conclusions Thanks to the entrapment into NLC systems, tyrphostin AG-1478 shows an enhanced in vitro anti-tumor activity compared to free drug. These finding raises hope of future drug delivery strategy of tyrphostin AG-1478 -loaded NLC targeted to the liver for the HCC treatment. PMID:24886097
The in vivo Therapeutic Effect of Free Wanderer Powder (逍遙散 xiāo yáo sǎn, Xiaoyaosan) on Mice with 4T1 Cell Induced Breast Cancer Model

PubMed Central

Chen, Wen-Fang; Xu, Li; Yu, Chung-Him; Ho, Chui-Kwan; Wu, Ka; Leung, Gina CW; Wong, Man-Sau

2012-01-01

In the present study, we investigated the therapeutic effect of a classical TCM formula, Free Wanderer Powder (逍遙散 xiāo yáo sǎn), in a breast cancer mouse model induced with estrogen-insensitive breast cancer 4T1 cells. Ovariectomized Balb/c mice (6-8 weeks) or sham mice were injected into the fourth mammary fat pad with 4T1 cells in which tumors were palpable 7 days after injection. On the eighth day, the mice were divided into 4 groups and tubefed daily with vehicle, Free Wanderer Powder (逍遙散 xiāo yáo sǎn) formula or tamoxifen for 28 days. Tumor growth inhibition and the decrease of the average tumor mass were most evident in mice treated with Free Wanderer Powder (逍遙散 xiāo yáo sǎn). Free Wanderer Powder (逍遙散 xiāo yáo sǎn) treatment significantly reduced Bcl-2 and elevated Bax and p53 protein expressions in breast cancer tumor. These results were further confirmed by immunohistochemisty. Tamoxifen could decrease spleen mass and Bcl-2 protein expression, increase the Bax protein expression as well as exert uterotrophic effects by increasing uterus index and inducing the gene expressions in the uterus. Taken together, these results show that Free Wanderer Powder (逍遙散 xiāo yáo sǎn) treatment induced apoptosis at protein level and inhibited the tumor growth in 4T1-induced ovariectomized Balb/c female mice, indicating the possibility of its future use for treatment of estrogen-insensitive breast caner. PMID:24716117
An integrated cell-free metabolic platform for protein production and synthetic biology

PubMed Central

Jewett, Michael C; Calhoun, Kara A; Voloshin, Alexei; Wuu, Jessica J; Swartz, James R

2008-01-01

Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements. PMID:18854819
Reduced beta 2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme.

PubMed Central

Larsson, S; Hotchkiss, G; Andäng, M; Nyholm, T; Inzunza, J; Jansson, I; Ahrlund-Richter, L

1994-01-01

We have generated three artificial hammerhead ribozymes, denoted 'Rz-b', 'Rz-c' and 'Rz-d', with different specificities for exon II of the mouse beta-2-microglobulin (beta 2M) mRNA. In this study we tested for ribozyme mediated reduction of beta 2M mRNA in a cell line and in transgenic mice. Transfections of either of the Rz-b, Rz-c or Rz-d plasmids into a mouse cell-line (NIH/3T3) revealed reductions of beta 2M mRNA substrate in each case. Ribozyme expression in individual transfected clones was accompanied with an up to 80% reduction of beta 2M mRNA levels. Rz-c was selected for a transgenic study. Seven Rz-c transgenic founder animals were identified from which three ribozyme expressing families were established and analysed. Expression of the ribozyme transgene was tested for and detected in lung, kidney and spleen. Expression was accompanied with reduction of the beta 2M mRNA levels of heterozygous (Rz+/-) animals compared to non-transgenic litter mates. The effect was most pronounced in lung with more than 90% beta 2M mRNA reduction in individual mice. In summary, expression of our ribozymes in a cell free system, in a cell-line and in transgenic mice were all accompanied with reductions of beta 2M mRNA levels. Images PMID:8036151
Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

PubMed Central

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF. PMID:26053554
Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

PubMed

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.
Homozygously deleted gene DACH1 regulates tumor-initiating activity of glioma cells

PubMed Central

Watanabe, Akira; Ogiwara, Hideki; Ehata, Shogo; Mukasa, Akitake; Ishikawa, Shumpei; Maeda, Daichi; Ueki, Keisuke; Ino, Yasushi; Todo, Tomoki; Yamada, Yasuhiro; Fukayama, Masashi; Saito, Nobuhito; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Loss or reduction in function of tumor suppressor genes contributes to tumorigenesis. Here, by allelic DNA copy number analysis using single-nucleotide polymorphism genotyping array and mass spectrometry, we report homozygous deletion in glioblastoma multiformes at chromosome 13q21, where DACH1 gene is located. We found decreased cell proliferation of a series of glioma cell lines by forced expression of DACH1. We then generated U87TR-Da glioma cells, where DACH1 expression could be activated by exposure of the cells to doxycycline. Both ex vivo cellular proliferation and in vivo growth of s.c. transplanted tumors in mice are reduced in U87TR-Da cells with DACH1 expression (U87-DACH1-high), compared with DACH1-nonexpressing U87TR-Da cells (U87-DACH1-low). U87-DACH1-low cells form spheroids with CD133 and Nestin expression in serum-free medium but U87-DACH1-high cells do not. Compared with spheroid-forming U87-DACH1-low cells, adherent U87-DACH1-high cells display lower tumorigenicity, indicating DACH1 decreases the number of tumor-initiating cells. Gene expression analysis and chromatin immunoprecipitation assay reveal that fibroblast growth factor 2 (FGF2/bFGF) is transcriptionally repressed by DACH1, especially in cells cultured in serum-free medium. Exogenous bFGF rescues spheroid-forming activity and tumorigenicity of the U87-DACH1-high cells, suggesting that loss of DACH1 increases the number of tumor-initiating cells through transcriptional activation of bFGF. These results illustrate that DACH1 is a distinctive tumor suppressor, which does not only suppress growth of tumor cells but also regulates bFGF-mediated tumor-initiating activity of glioma cells. PMID:21750150
Gravity-stimulated changes in auxin and invertase gene expression in maize pulvinal cells

NASA Technical Reports Server (NTRS)

Long, Joanne C.; Zhao, Wei; Rashotte, Aaron M.; Muday, Gloria K.; Huber, Steven C.; Brown, C. S. (Principal Investigator)

2002-01-01

Maize (Zea mays) stem gravitropism involves differential elongation of cells within a highly specialized region, the stem internodal pulvinus. In the present study, we investigated factors that control gravitropic responses in this system. In the graviresponding pulvinus, hexose sugars (D-Glc and D-Fru) accumulated asymmetrically across the pulvinus. This correlated well with an asymmetric increase in acid invertase activity across the pulvinus. Northern analyses revealed asymmetric induction of one maize acid invertase gene, Ivr2, consistent with transcriptional regulation by gravistimulation. Several lines of evidence indicated that auxin redistribution, as a result of polar auxin transport, is necessary for gravity-stimulated Ivr2 transcript accumulation and differential cell elongation across the maize pulvinus. First, the auxin transport inhibitor, N-1-naphthylphthalamic acid, inhibited gravistimulated curvature and Ivr2 transcript accumulation. Second, a transient gradient of free indole-3-acetic acid (IAA) across the pulvinus was apparent shortly after initiation of gravistimulation. This temporarily free IAA gradient appears to be important for differential cell elongation and Ivr2 transcript accumulation. This is based on the observation that N-1-naphthylphthalamic acid will not inhibit gravitropic responses when applied to pulvinus tissue after the free IAA gradient peak has occurred. Third, IAA alone can stimulate Ivr2 transcript accumulation in non-gravistimulated pulvini. The gravity- and IAA-stimulated increase in Ivr2 transcripts was sensitive to the protein synthesis inhibitor, cycloheximide. Based on these results, a two-phase model describing possible relationships between gravitropic curvature, IAA redistribution, and Ivr2 expression is presented.
Transcriptome Profiling of Bovine Milk Oligosaccharide Metabolism Genes Using RNA-Sequencing

PubMed Central

Wickramasinghe, Saumya; Hua, Serenus; Rincon, Gonzalo; Islas-Trejo, Alma; German, J. Bruce; Lebrilla, Carlito B.; Medrano, Juan F.

2011-01-01

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk. PMID:21541029
A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells.

PubMed

Stech, Marlitt; Quast, Robert B; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds.
A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

PubMed Central

Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975
Characterization of pancreatic stem cells derived from adult human pancreas ducts by fluorescence activated cell sorting.

PubMed

Lin, Han-Tso; Chiou, Shih-Hwa; Kao, Chung-Lan; Shyr, Yi-Ming; Hsu, Chien-Jen; Tarng, Yih-Wen; Ho, Larry L-T; Kwok, Ching-Fai; Ku, Hung-Hai

2006-07-28

To isolate putative pancreatic stem cells (PSCs) from human adult tissues of pancreas duct using serum-free, conditioned medium. The characterization of surface phenotype of these PSCs was analyzed by flow cytometry. The potential for pancreatic lineage and the capability of beta-cell differentiation in these PSCs were evaluated as well. By using serum-free medium supplemented with essential growth factors, we attempted to isolate the putative PSCs which has been reported to express nestin and pdx-1. The Matrigel(TM) was employed to evaluate the differential capacity of isolated cells. Dithizone staining, insulin content/secretion measurement, and immunohistochemistry staining were used to monitor the differentiation. Fluorescence activated cell sorting (FACS) was used to detect the phenotypic markers of putative PSCs. A monolayer of spindle-like cells was cultivated. The putative PSCs expressed pdx-1 and nestin. They were also able to differentiate into insulin-, glucagon-, and somatostatin-positive cells. The spectrum of phenotypic markers in PSCs was investigated; a similarity was revealed when using human bone marrow-derived stem cells as the comparative experiment, such as CD29, CD44, CD49, CD50, CD51, CD62E, PDGFR-alpha, CD73 (SH2), CD81, CD105(SH3). In this study, we successfully isolated PSCs from adult human pancreatic duct by using serum-free medium. These PSCs not only expressed nestin and pdx-1 but also exhibited markers attributable to mesenchymal stem cells. Although work is needed to elucidate the role of these cells, the application of these PSCs might be therapeutic strategies for diabetes mellitus.
Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures.

PubMed

Gebhard, C; Gabriel, C; Walter, I

2016-06-01

Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold-free three-dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19-day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14- and 19-day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki-67 immunoreactivity showed an even distribution in two-dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis-associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold-free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell-cell and cell-matrix interactions. © 2015 The Authors. Anatomia, Histologia, Embryologia Published by Blackwell Verlag GmbH.
A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.

PubMed

Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

2014-01-01

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.
A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

PubMed Central

Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N.; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao

2015-01-01

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10–50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. PMID:25742692

Mechanism of action of tetrandrine, a natural inhibitor of Candida albicans drug efflux pumps.

PubMed

Zhang, Hong; Gao, Aili; Li, Fengxia; Zhang, Gehua; Ho, Hon In; Liao, Wanqing

2009-05-01

Synergistic effects have previously been observed for a natural compound, tetrandrine (TET), with fluconazole (FLC) in vitro and in the treatment of Candida albicans-infected mice. To investigate the mechanisms of these synergistic effects, 16 strains of C. albicans from the same parent but with different FLC sensitivities were examined using flow cytometry and fluorescent spectrophotometry. Rhodamine 123 (Rh123)-positive cells and intracellular Rh123 fluorescence intensity were determined in accumulation/efflux experiments involving no or a noncytotoxic dose of TET. Total RNA extracted from each strain was used to compare the expressions of drug efflux pump genes in FLC-susceptible, -susceptible dose-dependent, and -resistant strains before and 24 h after TET administration. Accumulation experiments determined that mean percentages of Rh123-positive cells were 26.65% (TET-free) and 70.99% (TET 30 microg/ml), and mean respective intracellular Rh123 fluorescence intensities were 11.34 and 18.00. Efflux experiments showed that percentages of Rh123-positive cells were 1.79% (TET free) and 42.57% (TET 30 microg/ml), respectively, and respective mean intracellular Rh123 fluorescence intensities were 0.74 and 2.19. Differences in MDR1, FLU1, CDR1, and CDR2 expression levels in the absence of TET were statistically significant (p<0.05) between FLC-susceptible, -susceptible dose-dependent, and -resistant strains. Compared with TET-free conditions, 24 h TET-treated strains showed statistically different (p<0.05) expression of MDR1 (FLC-resistant strain), FLU1 (FLC-susceptible dose-dependent and -resistant strains), and CDR1 and CDR2 (FLC-susceptible, -susceptible dose-dependent, and -resistant strains). Thus TET can inhibit the C. albicans drug efflux system and reduce drug efflux. Its mechanism of action is related to the inhibition of expression of the drug efflux pump genes MDR1, FLU1, CDR1, and CDR2.
Secretion of autoimmune antibodies in the human subcutaneous adipose tissue

PubMed Central

Diaz, Alain; Romero, Maria; Thaller, Seth; Blomberg, Bonnie B.

2018-01-01

The adipose tissue (AT) contributes to systemic and B cell intrinsic inflammation, reduced B cell responses and secretion of autoimmune antibodies. In this study we show that adipocytes in the human obese subcutaneous AT (SAT) secrete several pro-inflammatory cytokines and chemokines, which contribute to the establishment and maintenance of local and systemic inflammation, and consequent suboptimal immune responses in obese individuals, as we have previously shown. We also show that pro-inflammatory chemokines recruit immune cells expressing the corresponding receptors to the SAT, where they also contribute to local and systemic inflammation, secreting additional pro-inflammatory mediators. Moreover, we show that the SAT generates autoimmune antibodies. During the development of obesity, reduced oxygen and consequent hypoxia and cell death lead to further release of pro-inflammatory cytokines, “self” protein antigens, cell-free DNA and lipids. All these stimulate class switch and the production of autoimmune IgG antibodies which have been described to be pathogenic. In addition to hypoxia, we have measured cell cytotoxicity and DNA damage mechanisms, which may also contribute to the release of “self” antigens in the SAT. All these processes are significantly elevated in the SAT as compared to the blood. We definitively found that fat-specific IgG antibodies are secreted by B cells in the SAT and that B cells express mRNA for the transcription factor T-bet and the membrane marker CD11c, both involved in the production of autoimmune IgG antibodies. Finally, the SAT also expresses RNA for cytokines known to promote Germinal Center formation, isotype class switch, and plasma cell differentiation. Our results show novel mechanisms for the generation of autoimmune antibody responses in the human SAT and allow the identification of new pathways to possibly manipulate in order to reduce systemic inflammation and autoantibody production in obese individuals. PMID:29768501
Secretion of autoimmune antibodies in the human subcutaneous adipose tissue.

PubMed

Frasca, Daniela; Diaz, Alain; Romero, Maria; Thaller, Seth; Blomberg, Bonnie B

2018-01-01

The adipose tissue (AT) contributes to systemic and B cell intrinsic inflammation, reduced B cell responses and secretion of autoimmune antibodies. In this study we show that adipocytes in the human obese subcutaneous AT (SAT) secrete several pro-inflammatory cytokines and chemokines, which contribute to the establishment and maintenance of local and systemic inflammation, and consequent suboptimal immune responses in obese individuals, as we have previously shown. We also show that pro-inflammatory chemokines recruit immune cells expressing the corresponding receptors to the SAT, where they also contribute to local and systemic inflammation, secreting additional pro-inflammatory mediators. Moreover, we show that the SAT generates autoimmune antibodies. During the development of obesity, reduced oxygen and consequent hypoxia and cell death lead to further release of pro-inflammatory cytokines, "self" protein antigens, cell-free DNA and lipids. All these stimulate class switch and the production of autoimmune IgG antibodies which have been described to be pathogenic. In addition to hypoxia, we have measured cell cytotoxicity and DNA damage mechanisms, which may also contribute to the release of "self" antigens in the SAT. All these processes are significantly elevated in the SAT as compared to the blood. We definitively found that fat-specific IgG antibodies are secreted by B cells in the SAT and that B cells express mRNA for the transcription factor T-bet and the membrane marker CD11c, both involved in the production of autoimmune IgG antibodies. Finally, the SAT also expresses RNA for cytokines known to promote Germinal Center formation, isotype class switch, and plasma cell differentiation. Our results show novel mechanisms for the generation of autoimmune antibody responses in the human SAT and allow the identification of new pathways to possibly manipulate in order to reduce systemic inflammation and autoantibody production in obese individuals.
Reconstituted High-Density Lipoprotein Containing Human Growth Hormone-1 Shows Potent Tissue Regeneration Activity with Enhancement of Anti-Oxidant and Anti-Atherosclerotic Activities.

PubMed

Lee, Eun-Young; Kim, So-Hee; Cho, Kyung-Hyun

2015-06-01

Human growth hormone-1 (GH-1), somatotropin, is a peptide hormone that stimulates cell division in tissues such as bone and cartilage. To compare physiological activities in lipid-free and lipid-bound states, we expressed and incorporated GH-1 in reconstituted high-density lipoprotein (rHDL). GH-1 was expressed and purified using the pET30(a) vector and an Escherichia coli expression system. Purified GH-1 (at least 98% purity, 23 kD) was characterized and synthesized with apolipoproteinA-I (apoA-I), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol. Secondary structure analysis of GH-1 revealed 54% α-helical content in a lipid-free state and 65% α-helical content in a lipid-bound state along with blue-shifted tryptophan movement (around 2 nm). GH-1 caused less uptake of oxidized low-density lipoprotein (oxLDL) into macrophages and inhibited senescence of dermal cells in a dose-dependent manner. GH-1 in a lipid-bound state exerted stronger inhibitory activity than in a lipid-free state, indicating improved anti-atherosclerotic activity due to the lipid formulation. In a fin regeneration experiment using zebrafish (17 weeks old, n=9), GH-1 showed its highest regeneration speed without any side effects. GH-1-rHDL containing apoA-I showed 2.3-fold and 1.6-fold higher regeneration speeds than lipid-free GH-1 (in native state) and lipid-bound GH-1, respectively. Incorporation of GH-1 and apoA-I in HDL enhanced tissue regeneration activity of amputated tail fin, indicating a synergetic effect between GH-1 and apoA-I in a lipid-bound state.
Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.

PubMed

Li, Jingsong; Luo, Ruihua; Hooi, Shing Chuan; Ruga, Pilar; Zhang, Jiping; Meda, Paolo; Li, GuoDong

2005-03-22

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.
Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

PubMed Central

Kebriaei, Partow; Singh, Harjeet; Huls, M. Helen; Figliola, Matthew J.; Bassett, Roland; Olivares, Simon; Jena, Bipulendu; Dawson, Margaret J.; Kumaresan, Pappanaicken R.; Su, Shihuang; Maiti, Sourindra; Dai, Jianliang; Moriarity, Branden; Forget, Marie-Andrée; Senyukov, Vladimir; Orozco, Aaron; Liu, Tingting; McCarty, Jessica; Jackson, Rineka N.; Moyes, Judy S.; Rondon, Gabriela; Qazilbash, Muzaffar; Ciurea, Stefan; Alousi, Amin; Nieto, Yago; Rezvani, Katy; Marin, David; Popat, Uday; Hosing, Chitra; Shpall, Elizabeth J.; Kantarjian, Hagop; Keating, Michael; Wierda, William; Do, Kim Anh; Largaespada, David A.; Lee, Dean A.; Hackett, Perry B.; Champlin, Richard E.; Cooper, Laurence J.N.

2016-01-01

BACKGROUND. T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS. T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS. SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS. CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION. Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING. National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details. PMID:27482888
Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells.

PubMed

Kebriaei, Partow; Singh, Harjeet; Huls, M Helen; Figliola, Matthew J; Bassett, Roland; Olivares, Simon; Jena, Bipulendu; Dawson, Margaret J; Kumaresan, Pappanaicken R; Su, Shihuang; Maiti, Sourindra; Dai, Jianliang; Moriarity, Branden; Forget, Marie-Andrée; Senyukov, Vladimir; Orozco, Aaron; Liu, Tingting; McCarty, Jessica; Jackson, Rineka N; Moyes, Judy S; Rondon, Gabriela; Qazilbash, Muzaffar; Ciurea, Stefan; Alousi, Amin; Nieto, Yago; Rezvani, Katy; Marin, David; Popat, Uday; Hosing, Chitra; Shpall, Elizabeth J; Kantarjian, Hagop; Keating, Michael; Wierda, William; Do, Kim Anh; Largaespada, David A; Lee, Dean A; Hackett, Perry B; Champlin, Richard E; Cooper, Laurence J N

2016-09-01

T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.
A biotin-triggered genetic switch in mammalian cells and mice.

PubMed

Weber, Wilfried; Lienhart, Cédric; Baba, Marie Daoud-El; Fussenegger, Martin

2009-03-01

Adjustable and reversible transgene expression systems enabling precise control of metabolic pathways and tunable production of specific target proteins have been essential for conditional reprogramming of mammalian cells to achieve progress in basic and applied bioengineering disciplines. Most of the currently available transgene control modalities have been designed to be responsive to clinically licensed pharmacologically active drugs which were expected to prevail in future clinical trials yet raised concerns about side effects when administered long term at subclinical doses. We have chosen vitamin H, also known as biotin, to control target gene transcription in mammalian cells in a potentially side effect-free manner. BirA, the Escherichia coli repressor of the biotin biosynthesis operon, was fused to the Herpes simplex transactivation domain to generate a biotin-dependent transactivator(BIT), which, in the presence of biotin, binds and activates chimeric target promoters (P(BIT)) harboring BirA-specific operator sites 5' of a minimal promoter. Biotin-inducible transgene expression was functional in a variety of rodent, monkey and human cell lines, showed excellent adjustability and reversibility in transgenic Chinese hamster ovary cell lines, provided precise product gene control in standard bioreactor cultures and enabled dose-dependent vitamin H control of a human glycoprotein in mice. The combination of a side effect-free inducer, precise and reversible transcription tunability and broad functionality in different cell types as well as in entire animals represents a unique asset for the use of biotin-inducible transgene control in future gene therapy, tissue engineering and biopharmaceutical manufacturing scenarios.
Folic Acid supplementation stimulates notch signaling and cell proliferation in embryonic neural stem cells.

PubMed

Liu, Huan; Huang, Guo-Wei; Zhang, Xu-Mei; Ren, Da-Lin; X Wilson, John

2010-09-01

The present study investigated the effect of folic acid supplementation on the Notch signaling pathway and cell proliferation in rat embryonic neural stem cells (NSCs). The NSCs were isolated from E14-16 rat brain and grown as neurospheres in serum-free suspension culture. Individual cultures were assigned to one of 3 treatment groups that differed according to the concentration of folic acid in the medium: Control (baseline folic acid concentration of 4 mg/l), low folic acid supplementation (4 mg/l above baseline, Folate-L) and high folic acid supplementation (40 mg/l above baseline, Folate-H). NSCs were identified by their expression of immunoreactive nestin and proliferating cells by incorporation of 5'bromo-2'deoxyuridine. Cell proliferation was also assessed by methyl thiazolyl tetrazolium assay. Notch signaling was analyzed by real-time PCR and western blot analyses of the expression of Notch1 and hairy and enhancer of split 5 (Hes5). Supplementation of NSCs with folic acid increased the mRNA and protein expression levels of Notch1 and Hes5. Folic acid supplementation also stimulated NSC proliferation dose-dependently. Embryonic NSCs respond to folic acid supplementation with increased Notch signaling and cell proliferation. This mechanism may mediate the effects of folic acid supplementation on neurogenesis in the embryonic nervous system.
LC-MS analysis of Hep-2 and Hek-293 cell lines treated with Brazilian red propolis reveals differences in protein expression.

PubMed

da Silva Frozza, Caroline O; da Silva Brum, Emyle; Alving, Anjali; Moura, Sidnei; Henriques, João A P; Roesch-Ely, Mariana

2016-08-01

Red propolis, an exclusive variety of propolis found in the northeast of Brazil has shown to present antitumour activity, among several other biological properties. This article aimed to help to evaluate the underlying molecular mechanisms of the potential anticancer effects of red propolis on tumour, Hep-2, and non-tumour cells, Hek-293. Differentially expressed proteins in human cell lines were identified through label-free quantitative MS-based proteomic platform, and cells were stained with Giemsa to show morphological changes. A total of 1336 and 773 proteins were identified for Hep-2 and Hek-293, respectively. Among the proteins here identified, 16 were regulated in the Hep-2 cell line and 04 proteins in the Hek-293 line. Over a total of 2000 proteins were identified under MS analysis, and approximately 1% presented differential expression patterns. The GO annotation using Protein Analysis THrough Evolutionary Relationships classification system revealed predominant molecular function of catalytic activity, and among the biological processes, the most prominent was associated to cell metabolism. The proteomic profile here presented should help to elucidate further molecular mechanisms involved in inhibition of cancer cell proliferation by red propolis, which remain unclear to date. © 2016 Royal Pharmaceutical Society.
Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.
Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251
Derivation of Xeno-Free and GMP-Grade Human Embryonic Stem Cells – Platforms for Future Clinical Applications

PubMed Central

Aizenman, Einat; Kirshberg, Sophie; Ilouz, Nili; Gil, Yaniv; Berman-Zaken, Yael; Perlman, Temima Schnitzer; Geva, Nitshia; Levy, Ora; Arbell, Daniel; Simon, Alex; Ben-Meir, Assaf; Shufaro, Yoel; Laufer, Neri; Reubinoff, Benjamin E.

2012-01-01

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs. PMID:22745653
Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

PubMed

Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

2012-08-01

The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.
Feeder-free reprogramming of human fibroblasts with messenger RNA.

PubMed

Warren, Luigi; Wang, Jiwu

2013-11-13

This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.
Graphene quantum dots for cancer targeted drug delivery.

PubMed

Iannazzo, Daniela; Pistone, Alessandro; Salamò, Marina; Galvagno, Signorino; Romeo, Roberto; Giofré, Salvatore V; Branca, Caterina; Visalli, Giuseppa; Di Pietro, Angela

2017-02-25

A biocompatible and cell traceable drug delivery system Graphene Quantum Dots (GQD) based, for the targeted delivery of the DNA intercalating drug doxorubicin (DOX) to cancer cells, is here reported. Highly dispersible and water soluble GQD, synthesized by acidic oxidation and exfoliation of multi-walled carbon nanotubes (MWCNT), were covalently linked to the tumor targeting module biotin (BTN), able to efficiently recognize biotin receptors over-expressed on cancer cells and loaded with DOX. Biological test performed on A549 cells reported a very low toxicity of the synthesized carrier (GQD and GQD-BTN). In GQD-BTN-DOX treated cancer cells, the cytotoxicity was strongly dependent from cell uptake which was greater and delayed after treatment with GQD-BTN-DOX system with respect to what observed for cells treated with the same system lacking of the targeting module BTN (GQD-DOX) or with the free drug alone. A delayed nuclear internalization of the drug is reported, due to the drug detachment from the nanosystem, triggered by the acidic environment of cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.
Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

PubMed

Green, David W; Kim, Eun-Jung; Jung, Han-Sung

2015-09-01

The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. © 2015 Wiley Periodicals, Inc.
Characterization of fibroblast-free CWR-R1ca castration-recurrent prostate cancer cell line.

PubMed

Shourideh, Mojgan; DePriest, Adam; Mohler, James L; Wilson, Elizabeth M; Koochekpour, Shahriar

2016-09-01

The previously established CWR-R1 cell line has been used as an in vitro model representing castration-recurrent prostate cancer. Microscopic observation of subconfluent cells demonstrated two distinct cellular morphologies: polygonal closely aggregated epithelial cells surrounded by bipolar fibroblastic cells with long processes. This study sought to establish and characterize a fibroblast-free derivative of the CWR-R1 cell line. The CWR-R1ca cell line was established from CWR-R1 cells by removing fibroblasts using multiple cycles of short-term trypsinization, cloning, and pooling single-cell colonies. Authentication of fibroblast-free CWR-R1ca cells was demonstrated by analyzing the expression of cytodifferentiation and prostate-associated markers, DNA and cytogenetic profiling, and growth pattern in the absence or presence of androgen. CWR-R1ca is an androgen-sensitive cell line that expresses the androgen receptor (AR) and its splice variant 7 and the luminal epithelia markers, CK-8, CK-18, and c-Met. CWR-R1fb fibroblasts isolated from CWR-R1 cells express AR, hepatocyte growth factor-α, and mouse β-actin but not AR-V7 or epithelial markers. Cytogenetic analysis of CWR-R1ca cells revealed a hyperdiploid male with numerical gains in chromosomes 1, 7, 8, 10, 11, and 12, deletion of one chromosome 2 allele, structural abnormalities that include der(1)t(1:4), der(4)t(2:4), der(10)t(4:10), and an unbalanced reciprocal translocation between chromosome 6 and 14. DNA-profiling revealed that CWR-R1ca cells had significant short-tandem repeat marker homology with CWR22Pc and CWR22Rv1 cell lines, which indicated lineage derivation from CWR22 prostate cancer xenografts. CWR-R1ca cells were responsive to the growth stimulatory effects of dihydrotestosterone (DHT) in the femtomolar range. This study establishes CWR-R1ca cells as a fibroblast-free derivative of the castration-recurrent CWR-R1 cell line. Prostate 76:1067-1077, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Adjuvant properties of thermal component of hyperthermia enhanced transdermal immunization: effect on dendritic cells.

PubMed

Joshi, Neha; Duhan, Vikas; Lingwal, Neelam; Bhaskar, Sangeeta; Upadhyay, Pramod

2012-01-01

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential.
Enrichment of prostate cancer stem cells from primary prostate cancer cultures of biopsy samples

PubMed Central

Wang, Shunqi; Huang, Shengsong; Zhao, Xin; Zhang, Qimin; Wu, Min; Sun, Feng; Han, Gang; Wu, Denglong

2014-01-01

This study was to enrich prostate cancer stem cells (PrCSC) from primary prostate cancer cultures (PPrCC). Primary prostate cancer cells were amplified in keratinocyte serum-free medium with epidermal growth factor (EGF) and bovine pituitary extract (BPE), supplemented with leukemia inhibitory factor (LIF), stem cell factor (SCF) and cholera toxin. After amplification, cells were transferred into ultra-low attachment dishes with serum-free DMEM/F12 medium, supplemented with EGF, basic fibroblast growth factor (bFGF), bovine serum albumin (BSA), insulin, and N2 nutrition. Expression of cell-type-specific markers was determined by RT-qPCR and immunostaining. Tumorigenicity of enriched PrCSC was determined by soft agar assay and xenograft assay in NOD/SCID mice. Biopsy samples from 19 confirmed prostate cancer patients were used for establishing PPrCC, and 18 cases (95%) succeeded. Both basal marker (CK5) and luminal markers (androgen receptor and CK8) strongly co-expressed in most of PPrCC, indicating their basal epithelial origin. After amplification under adherent culture condition in vitro, transient amplifying cells were the dominant cells. Sphere formation efficiency (SFE) of passaged PPrCC was about 0.5%, which was 27 times lower than SFE of LNCaP (13.67%) in the same condition. Compared with adherent cells from PPrCC, prostasphere from PPrCC showed up regulated stem cell markers and increased tumorigenic potential in soft-agar assay. However, spheroid cells from PPrCC prostasphere failed to initiate tumor in xenograft assay in 6 months. Thus, PPrCC can be established and amplified from prostate cancer biopsy samples. Our modified sphere culture system can enrich PrCSC from PPrCC. PMID:24427338

p62/SQSTM1 interacts with vimentin to enhance breast cancer metastasis.

PubMed

Li, Si-Si; Xu, Ling-Zhi; Zhou, Wei; Yao, Shang; Wang, Chun-Li; Xia, Jiang-Long; Wang, He-Fei; Kamran, Muhammad; Xue, Xiao-Yuan; Dong, Lin; Wang, Jing; Ding, Xu-Dong; Bella, Laura; Bugeon, Laurence; Xu, Jie; Zheng, Fei-Meng; Dallman, Margaret J; Lam, Eric W F; Liu, Quentin

2017-10-26

The signalling adaptor p62 is frequently overexpressed in numerous cancer types. Here, we found that p62 expression was elevated in metastatic breast cancer and its overexpression correlated with reduced metastasis- and relapse-free survival times. Analysis of p62 expression in breast cancer cell lines demonstrated that high p62 expression was associated with the invasive phenotypes of breast cancer. Indeed, silencing p62 expression attenuated the invasive phenotypes of highly metastatic cells, whereas overexpressing p62 promoted the invasion of non-metastatic cells in in vitro microfluidic model. Moreover, MDA-MB-231 cells with p62 depletion which were grown in a three-dimensional culture system exhibited a loss of invasive protrusions. Consistently, genetic ablation of p62 suppressed breast cancer metastasis in both zebrafish embryo and immunodeficient mouse models, as well as decreased tumourigenicity in vivo. To explore the molecular mechanism by which p62 promotes breast cancer invasion, we performed a co-immunoprecipitation-mass spectrometry analysis and revealed that p62 interacted with vimentin, which mediated the function of p62 in promoting breast cancer invasion. Vimentin protein expression was downregulated upon p62 suppression and upregulated with p62 overexpression in breast cancer cells. Linear regression analysis of clinical breast cancer specimens showed a positive correlation between p62 and vimentin protein expression. Together, our findings provide strong evidence that p62 functions as a tumour metastasis promoter by binding vimentin and promoting its expression. This finding might help to develop novel molecular therapeutic strategies for breast cancer metastasis treatment. © The Author 2017. Published by Oxford University Press.
Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

USDA-ARS?s Scientific Manuscript database

Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose employing Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuo...
Protonation free energy levels in complex molecular systems.

PubMed

Antosiewicz, Jan M

2008-04-01

All proteins, nucleic acids, and other biomolecules contain residues capable of exchanging protons with their environment. These proton transfer phenomena lead to pH sensitivity of many molecular processes underlying biological phenomena. In the course of biological evolution, Nature has invented some mechanisms to use pH gradients to regulate biomolecular processes inside cells or in interstitial fluids. Therefore, an ability to model protonation equilibria in molecular systems accurately would be of enormous value for our understanding of biological processes and for possible rational influence on them, like in developing pH dependent drugs to treat particular diseases. This work presents a derivation, by thermodynamic and statistical mechanical methods, of an expression for the free energy of a complex molecular system at arbitrary ionization state of its titratable residues. This constitutes one of the elements of modeling protonation equilibria. Starting from a consideration of a simple acid-base equilibrium of a model compound with a single tritratable group, we arrive at an expression which is of general validity for complex systems. The only approximation used in this derivation is the postulating that the interaction energy between any pair of titratable sites does not depend on the protonation states of all the remaining ionizable groups.
Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

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

Yin, Xiaohui; Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191; Li, Yang

Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintainedmore » a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.« less
Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

PubMed

Lin, Juqiang; Xu, Han; Wu, Yangzhe; Tang, Mingjie; McEwen, Gerald D; Liu, Pin; Hansen, Dane R; Gilbertson, Timothy A; Zhou, Anhong

2013-02-05

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.
ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma.

PubMed

Dréan, Antonin; Rosenberg, Shai; Lejeune, François-Xavier; Goli, Larissa; Nadaradjane, Aravindan Arun; Guehennec, Jérémy; Schmitt, Charlotte; Verreault, Maïté; Bielle, Franck; Mokhtari, Karima; Sanson, Marc; Carpentier, Alexandre; Delattre, Jean-Yves; Idbaih, Ahmed

2018-03-08

ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p < 0.01) and multivariate analyses including MGMT promoter methylation (p = 0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM-PDCL. ABCA13 expression is an independent prognostic factor in newly diagnosed GBM patients. Further prospective studies are warranted to investigate whether ABCA13 expression can be used to further personalize treatments for GBM.
Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

PubMed Central

Ilinskaya, Anna; Dorjbal, Batsukh; Truong, Rosaline; Derse, David; Uchil, Pradeep D.; Heidecker, Gisela; Mothes, Walther

2013-01-01

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV. PMID:23308151
Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method.

PubMed

Ohnishi, Hiroe; Skerleva, Desislava; Kitajiri, Shin-ichiro; Sakamoto, Tatsunori; Yamamoto, Norio; Ito, Juichi; Nakagawa, Takayuki

2015-07-10

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic fibroblast growth factor (bFGF) for induction of differentiation into otic-placode-like cells for 15 days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells.

PubMed

Kamath, Anant; Ternes, Sara; McGowan, Stephen; English, Anthony; Mallampalli, Rama; Moy, Alan B

2017-08-01

Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28 . We describe a robust Myc and Lin28 -free IPSC reprogramming approach using reprogramming molecules. IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28 -free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28 . This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.
Hydrogen gas protects against serum and glucose deprivation‑induced myocardial injury in H9c2 cells through activation of the NF‑E2‑related factor 2/heme oxygenase 1 signaling pathway.

PubMed

Xie, Qiang; Li, Xue-Xiang; Zhang, Peng; Li, Jin-Cao; Cheng, Ying; Feng, Yan-Ling; Huang, Bing-Sheng; Zhuo, Yu-Feng; Xu, Guo-Hua

2014-08-01

Ischemia or hypoxia‑induced myocardial injury is closely associated with oxidative stress. Scavenging free radicals and/or enhancing endogenous antioxidative defense systems may be beneficial for the impediment of myocardial ischemic injury. Hydrogen (H2) gas, as a water‑ and lipid‑soluble small molecule, is not only able to selectively eliminate hydroxyl (·OH) free radicals, but also to enhance endogenous antioxidative defense systems in rat lungs and arabidopsis plants. However, thus far, it has remained elusive whether H2 gas protects cardiomyocytes through enhancement of endogenous antioxidative defense systems. In the present study, the cardioprotective effect of H2 gas against ischemic or hypoxic injury was investigated, along with the underlying molecular mechanisms. H9c2 cardiomyoblasts (H9c2 cells) were treated in vitro with a chemical hypoxia inducer, cobalt chloride (CoCl2), to imitate hypoxia, or by serum and glucose deprivation (SGD) to imitate ischemia. Cell viability and intracellular ·OH free radicals were assessed. The role of an endogenous antioxidative defense system, the NF‑E2‑related factor 2 (Nrf2)/heme oxygenase 1 (HO‑1) signaling pathway, was evaluated. The findings revealed that treatment with CoCl2 or SGD markedly reduced cell viability in H9c2 cells. H2 gas‑rich medium protected against cell injury induced by SGD, but not that induced by CoCl2. When the cells were exposed to SGD, levels of intracellular ·OH free radicals were markedly increased; this was mitigated by H2 gas‑rich medium. Exposure of the cells to SGD also resulted in significant increases in HO‑1 expression and nuclear Nrf2 levels, and the HO‑1 inhibitor ZnPP IX and the Nrf2 inhibitor brusatol aggravated SGD‑induced cellular injury. H2 gas‑rich medium enhanced SGD‑induced upregulation of HO‑1 and Nrf2, and the HO‑1 or Nrf2 inhibition partially suppressed H2 gas‑induced cardioprotection. Furthermore, following genetic silencing of Nrf2 by RNA interference, the effects of H2 gas on the induction of HO‑1 and cardioprotection were markedly reduced. In conclusion, H2 gas protected cardiomyocytes from ischemia‑induced myocardial injury through elimination of ·OH free radicals and also through activation of the Nrf2/HO‑1 signaling pathway.
Effect of Storage Temperature on the Phenotype of Cultured Epidermal Cells Stored in Xenobiotic-Free Medium.

PubMed

Jackson, Catherine; Eidet, Jon R; Reppe, Sjur; Aass, Hans Christian D; Tønseth, Kim A; Roald, Borghild; Lyberg, Torstein; Utheim, Tor P

2016-06-01

Cultured epidermal cell sheets (CECS) are used in the treatment of large area burns to the body and have potential to treat limbal stem cell deficiency (LSCD) as shown in animal studies. Despite widespread use, storage options for CECS are limited. Short-term storage allows flexibility in scheduling surgery, quality control and improved transportation to clinics worldwide. Recent evidence points to the phenotype of cultured epithelial cells as a critical predictor of post-operative success following transplantation of CECS in burns and in transplantation of cultured epithelial cells in patients with LSCD. This study, therefore assessed the effect of a range of temperatures, spanning 4-37 °C, on the phenotype of CECS stored over a 2-week period in a xenobiotic-free system. Progenitor cell (p63, ΔNp63α and ABCG2) and differentiation (C/EBPδ and CK10) associated marker expression was assessed using immunocytochemistry. Immunohistochemistry staining of normal skin for the markers p63, ABCG2 and C/EBPδ was also carried out. Assessment of progenitor cell side population (SP) was performed using JC1 dye by flow cytometry. P63 expression remained relatively constant throughout the temperature range but was significantly lower compared to control between 20 and 28 °C (p < 0.05). High C/EBPδ together with low p63 suggested more differentiation beginning at 20 °C and above. Lower CK10 and C/EBPδ expression most similar to control was seen at 12 °C. The percentage of ABCG2 positive cells was most similar to control between 8 and 24 °C. Between 4 and 24 °C, the SP fluctuated, but was not significantly different compared to control. Results were supported by staining patterns indicating differentiation status associated with markers in normal skin sections. Lower storage temperatures, and in particular 12 °C, merit further investigation as optimal storage temperature for maintenance of undifferentiated phenotype in CECS.
Difference in suitable mechanical properties of three-dimensional, synthetic scaffolds for self-renewing mouse embryonic stem cells of different genetic backgrounds.

PubMed

Lee, Myungook; Ahn, Jong Il; Ahn, Ji Yeon; Yang, Woo Sub; Hubbell, Jeffrey A; Lim, Jeong Mook; Lee, Seung Tae

2017-11-01

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p < 0.0001) was observed in the four-arm 7.5% PEG-based hydrogels than those with other properties, whereas the F1 ESCs showed better proliferation when they were embedded in the three-arm 10% hydrogels. Self-renewal-related gene and protein expression by ESCs after feeder-free 3D culture was generally maintained compared with the feeder-containing 2D culture, but expression patterns and quantities differed. However, the feeder-free 3D culture yielded better expression than the feeder-free 2D culture. In conclusion, genetic background determined the suitability of hydrogel scaffolds for self-renewal of ESCs, which requires customization for the mechanical properties of each cell line. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2261-2268, 2017. © 2016 Wiley Periodicals, Inc.
Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells.

PubMed

Qiu, Caihong; Olivier, Emmanuel N; Velho, Michelle; Bouhassira, Eric E

2008-02-15

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA.
Effect of fraxetin on antioxidant defense and stress proteins in human neuroblastoma cell model of rotenone neurotoxicity. Comparative study with myricetin and N-acetylcysteine

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

Molina-Jimenez, Maria Francisca; Sanchez-Reus, Maria Isabel; Cascales, Maria

2005-12-15

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. Recently, it has been shown that fraxetin (coumarin) and myricetin (flavonoid) have significant neuroprotective effects against apoptosis induced by rotenone, increase the total glutathione levels in vitro, and inhibit lipid peroxidation. Thus, these considerations prompted us to investigate the way in which fraxetin and myricetin affect the endogenous antioxidant defense system, such as Mn and CuZn superoxide dismutase (MnSOD, CuZnSOD), catalase, glutathione reductase (GR), and glutathione peroxidase (GPx) on rotenone neurotoxicity in neuroblastoma cells. N-acetylcysteine (NAC), a potent antioxidant, was employed as a comparative agent. Also,more » the expression and protein levels of HSP70 by Northern and Western blot analysis were assayed in SH-SY5Y cells. After incubation for 16 h, rotenone significantly increased the expression and activity of MnSOD, GPx, and catalase. When cells were preincubated with fraxetin, there was a decrease in the protein levels and activity of both MnSOD and catalase, in comparison with the rotenone treatment. The myricetin effect was less pronounced. Activity and expression of GPx were increased by rotenone and pre-treatment with fraxetin did not modify significantly these levels. The significant enhancement in HSP70 expression at mRNA and protein levels induced by fraxetin was observed by pre-treatment of cells 0.5 h before rotenone insult. These data suggest that major features of rotenone-induced neurotoxicity are partially mediated by free radical formation and oxidative stress, and that fraxetin partially protects against rotenone toxicity affecting the main protection system of the cells against oxidative injury.« less
Functional Dissection of Sugar Signals Affecting Gene Expression in Arabidopsis thaliana

PubMed Central

Kunz, Sabine; Pesquet, Edouard; Kleczkowski, Leszek A.

2014-01-01

Background Sugars modulate expression of hundreds of genes in plants. Previous studies on sugar signaling, using intact plants or plant tissues, were hampered by tissue heterogeneity, uneven sugar transport and/or inter-conversions of the applied sugars. This, in turn, could obscure the identity of a specific sugar that acts as a signal affecting expression of given gene in a given tissue or cell-type. Methodology/Principal Findings To bypass those biases, we have developed a novel biological system, based on stem-cell-like Arabidopsis suspension culture. The cells were grown in a hormone-free medium and were sustained on xylose as the only carbon source. Using functional genomics we have identified 290 sugar responsive genes, responding rapidly (within 1 h) and specifically to low concentration (1 mM) of glucose, fructose and/or sucrose. For selected genes, the true nature of the signaling sugar molecules and sites of sugar perception were further clarified using non-metabolizable sugar analogues. Using both transgenic and wild-type A. thaliana seedlings, it was shown that the expression of selected sugar-responsive genes was not restricted to a specific tissue or cell type and responded to photoperiod-related changes in sugar availability. This suggested that sugar-responsiveness of genes identified in the cell culture system was not biased toward heterotrophic background and resembled that in whole plants. Conclusions Altogether, our research strategy, using a combination of cell culture and whole plants, has provided an unequivocal evidence for the identity of sugar-responsive genes and the identity of the sugar signaling molecules, independently from their inter-conversions or use for energy metabolism. PMID:24950222
Role of Indigenous Lactobacilli in Gastrin-Mediated Acid Production in the Mouse Stomach ▿

PubMed Central

Takahashi, Hidenori; Nakano, Yasuhiro; Matsuoka, Takashi; Kumaki, Nobue; Asami, Yukio; Koga, Yasuhiro

2011-01-01

It is known that the stomach is colonized by indigenous lactobacilli in mice. The aim of this study was to examine the role of such lactobacilli in the development of the stomach. For a DNA microarray analysis, germ-free BALB/c mice were orally inoculated with 109 CFU lactobacilli, and their stomachs were excised after 10 days to extract RNA. As a result, lactobacillus-associated gnotobiotic mice showed dramatically decreased expression of the gastrin gene in comparison to germ-free mice. The mean of the log2 fold change in the gastrin gene was −4.3. Immunohistochemistry also demonstrated the number of gastrin-positive (gastrin+) cells to be significantly lower in the lactobacillus-associated gnotobiotic mice than in the germ-free mice. However, there was no significant difference in the number of somatostatin+ cells in these groups of mice. Consequently, gastric acid secretion also decreased in the mice colonized by lactobacilli. In addition, an increase in the expression of the genes related to muscle system development, such as nebulin and troponin genes, was observed in lactobacillus-associated mice. Moreover, infection of germ-free mice with Helicobacter pylori also showed the down- and upregulation of gastrin and muscle genes, respectively, in the stomach. These results thus suggested that indigenous lactobacilli in the stomach significantly affect the regulation of gastrin-mediated gastric acid secretion without affecting somatostatin secretion in mice, while H. pylori also exerts such an effect on the stomach. PMID:21803885
Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

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

Yue, Xiao-shan; Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501; Fujishiro, Masako

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells weremore » tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.« less
Modulation of ColE1-like Plasmid Replication for Recombinant Gene Expression

PubMed Central

Camps, Manel

2010-01-01

ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of R-loop formation, and of the timing of recombinant gene expression. PMID:20218961
Expression and prognostic value of hemopoietic cytokine receptors in acute myeloid leukemia (AML): implications for future therapeutical strategies.

PubMed

Graf, Michaela; Hecht, Karin; Reif, Susanne; Pelka-Fleischer, Renate; Pfister, Karin; Schmetzer, Helga

2004-02-01

Hemopoietic cytokines regulate hemopoietic cell functions via specific cell surface receptors. There is evidence to suggest, that those receptors (R) could play a role in leukemia with respect to cell differentiations and its regulation, prognosis, and pathobiology. Knowledge of individual cytokine receptor (CKR) profiles could provide new discoveries about CKR-supported therapeutic considerations. We have studied the expression of CKR on mononuclear bone marrow (BM) cells of 89 patients with acute myeloid leukemia (AML) at first diagnosis, three patients at relapse or with persisting AML and eight healthy probands by fluorescence-activated cell sorting (FACS) analysis using directly fluorescein-conjugated antibodies: CD114 (hG-CSF-R), CD116 (hGM-CSF-R), CD117 (hSCF-R), CD123 (hIL-3-R), CD130 (gp130subunit), CD135 (hFL-R). A case was defined as positive, if more than 20% of the cells expressed the regarding CKR. All investigated CKR were more frequently expressed in AML-samples than in healthy BM-samples, except CD130, which was only expressed on 5-6% of AML-blasts in all and with only one healthy BM-sample being CD130(+). Within the French-American-British (FAB) types we observed a maturation- and lineage (granulocytic/monocytic)-committed expression profile. Monocytic subtypes (FAB-type M4/M5) showed significantly more GM-CSF-R(+) (P = 0.001) and FL-R(+) (P = 0.001) and significantly less stem cell factor-R (SCF-R(+)) (P = 0.02) cases. Highest proportions of G-CSF-R(+) blasts were observed in FAB-type M3. In undifferentiated leukemias (FAB-type M1, M2) high amounts of SCF-R(+), IL-3-R(+), and FL-R(+) blasts could be detected. FL-R was the only CKR, which was positive in FAB-type M0 (n = 2). No differences in CKR-expression were detected between primary (p) and secondary (s). Separating our patient cohorts in cytogenetic risk groups we could detect a significant higher proportion of G-CSF-R(+) blasts in the cytogenetic good risk group than in the bad risk group (P = 0.027), but G-CSF-R-expression did not correlate with remission-rate or relapse-free survival probability of the patients. For clinical evaluation only patients treated by the AML-CG-protocol, were included (n = 53). There were no differences of CKR-expression in the responder and non-responder group, however, significant lower relapse-free survival probabilities for patients with more than 85.5% FL-R(+) (P = 0.001) and more than 45.5% SCF-R(+) blasts were found (P = 0.02). Patients with more than 32.5% IL-3-R(+) cells also showed a tendency to a lower relapse-free survival probability (P = 0.26), whereas patients with more than 33% GM-CSF-R(+) (P = 0.06) and patients with more than 52% G-CSF-R(+) (P = 0.175) blasts tended to have a higher relapse-free survival probability. We can conclude, that CKR-expression in AML is maturation- and lineage-committed and the proportions of especially early acting CKR have influence on relapse-free survival probability of AML-patients, independently of the karyotype. With respect to the individual CKR status the benefit of cytokines as priming agents, as agents to treat neutropenia or to influence the metabolism of chemotherapy can be discussed under new points of view.
Expression of neurokinin B/NK3 receptor and kisspeptin/KISS1 receptor in human granulosa cells.

PubMed

García-Ortega, J; Pinto, F M; Fernández-Sánchez, M; Prados, N; Cejudo-Román, A; Almeida, T A; Hernández, M; Romero, M; Tena-Sempere, M; Candenas, L

2014-12-01

Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells? The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells. The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown. This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus-oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT-PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca(2+) levels, [Ca(2+)]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured. NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca(2+)]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca(2+)]i. However, the [Ca(2+)]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB. The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells. Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells. This work was supported by grants from Ministerio de Economía y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucía (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System.

PubMed

Low, Karen; Wong, Lauren Y; Maldonado, Maricela; Manjunath, Chetas; Horner, Christopher B; Perez, Mark; Myung, Nosang V; Nam, Jin

2017-05-09

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study, a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time, in relation to cellular activities during self-renewal or lineage-specific differentiation, in a non-destructive, label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal, or subjected to mesendodermal or ectodermal differentiation, and correlating them to morphological (size, shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall, the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Evaluation of cannabinoid CB1 and CB2 receptors expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients' survival.

PubMed

Theocharis, Stamatios; Giaginis, Constantinos; Alexandrou, Paraskevi; Rodriguez, Jose; Tasoulas, Jason; Danas, Eugene; Patsouris, Efstratios; Klijanienko, Jerzy

2016-03-01

Cannabinoid receptors (CB1R and CB2R) constitute essential members of the endocannabinoid system (ECS) which participates in many different functions indispensable to homeostatic regulation in several tissues, exerting also antitumorigenic effects. The present study aimed to assess the clinical significance of CB1R and CB2R protein expression in mobile tongue squamous cell carcinoma (SCC). CB1R and CB2R expression was assessed immunohistochemically on 28 mobile tongue SCC tissue samples and was analyzed in relation with clinicopathological characteristics and overall and disease-free patients' survival. CB1R, CB2R, and concomitant CB1R/CB2R expression was significantly increased in older compared to younger mobile tongue SCC patients (p = 0.0243, p = 0.0079, and p = 0.0366, respectively). Enhanced CB2R and concomitant CB1R/CB2R expression was significantly more frequently observed in female compared to male mobile tongue SCC patients (p = 0.0025 and p = 0.0016, respectively). Elevated CB2R expression was significantly more frequently observed in mobile tongue SCC patients presenting well-defined tumor shape compared to those with diffuse (p = 0.0430). Mobile tongue SCC patients presenting enhanced CB1R, CB2R, or concomitant CB1R/CB2R expression showed significantly longer overall (log-rank test, p = 0.004, p = 0.011, p = 0.018, respectively) and disease-free (log-rank test, p = 0.003, p = 0.007, p = 0.027, respectively) survival times compared to those with low expression. In multivariate analysis, CB1R was identified as an independent prognostic factor for disease-free patients' survival (Cox-regression analysis, p = 0.032). The present study provides evidence that CB1R and CB2R may play a role in the pathophysiological aspects of the mobile tongue SCC and even each molecule may constitute a potential target for the development of novel anti-cancer drugs for this type of malignancy.
Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.

PubMed

Huang, Xiaosong; Wang, Ying; Yan, Wei; Smith, Cory; Ye, Zhaohui; Wang, Jing; Gao, Yongxing; Mendelsohn, Laurel; Cheng, Linzhao

2015-05-01

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479. © 2015 AlphaMed Press.
Mitochondrial ribosomal protein S18-2 is highly expressed in endometrial cancers along with free E2F1

PubMed Central

Iurchenko, Natalia; Kovalevska, Larysa; Stip, Maria C; Budnikova, Daria; Andersson, Sonia; Polischuk, Ludmila; Buchynska, Lubov; Kashuba, Elena

2016-01-01

Endometrial cancer (EC) is one of the most frequent causes of cancer death among women in developed countries. Histopathological diagnosis and imaging techniques for EC are limited, thus new prognostic markers are needed to offer patients the best treatment and follow-up. In the present paper we showed that the level of mitochondrial ribosomal protein MRPS18-2 (S18-2) increased in EC compared with the normal endometrium and hyperplasia, based on a study of 42 patient biopsies. Importantly, high expression of free E2F1 in EC correlates well with high S18-2 expression. The EC cell line HEC-1-A, which overexpresses S18-2 constitutively, showed an increased proliferation capacity in vitro and in vivo (in SCID mice). Moreover, pan-keratin, beta-catenin and E-cadherin signals are diminished in these cells, compared to the parental HEC-1-A line, in contrast to vimentin signal that is increased. This may be associated with epithelial-mesenchymal cell transition (EMT). We conclude that high expression of S18-2 and free E2F1, and low pan-keratin, beta-catenin, and E-cadherin signals might be a good set of prognostic markers for EC. PMID:26959119
One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system.

PubMed

Wattanapanitch, Methichit; Damkham, Nattaya; Potirat, Ponthip; Trakarnsanga, Kongtana; Janan, Montira; U-Pratya, Yaowalak; Kheolamai, Pakpoom; Klincumhom, Nuttha; Issaragrisil, Surapol

2018-02-26

Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future.
Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells.

PubMed

Chang, Chih-Wei; Su, Yu-Chin; Her, Guor-Mour; Ken, Chuian-Fu; Hong, Jiann-Ruey

2011-01-01

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12-24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24-48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection. Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis.
Humoral Activity of Cord Blood-Derived Stem/Progenitor Cells: Implications for Stem Cell-Based Adjuvant Therapy of Neurodegenerative Disorders

PubMed Central

Paczkowska, Edyta; Kaczyńska, Katarzyna; Pius-Sadowska, Ewa; Rogińska, Dorota; Kawa, Miłosz; Ustianowski, Przemysław; Safranow, Krzysztof; Celewicz, Zbigniew; Machaliński, Bogusław

2013-01-01

Background Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34+, and CD133+ cells, with that in unsorted, nucleated cells (NCs). Methods and Results The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34+, and CD133+ cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34+, and CD133+ cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34+ or CD133+ cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation. Conclusions Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders. PMID:24391835
Humoral activity of cord blood-derived stem/progenitor cells: implications for stem cell-based adjuvant therapy of neurodegenerative disorders.

PubMed

Paczkowska, Edyta; Kaczyńska, Katarzyna; Pius-Sadowska, Ewa; Rogińska, Dorota; Kawa, Miłosz; Ustianowski, Przemysław; Safranow, Krzysztof; Celewicz, Zbigniew; Machaliński, Bogusław

2013-01-01

Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34(+), and CD133(+) cells, with that in unsorted, nucleated cells (NCs). The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34(+), and CD133(+) cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34(+), and CD133(+) cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34(+) or CD133(+) cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation. Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders.
Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

PubMed Central

Quast, Robert B.; Ballion, Biljana; Stech, Marlitt; Sonnabend, Andrei; Varga, Balázs R.; Wüstenhagen, Doreen A.; Kele, Péter; Schiller, Stefan M.; Kubick, Stefan

2016-01-01

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system. PMID:27670253
Rasagiline protects against alpha-synuclein induced sensitivity to oxidative stress in dopaminergic cells

PubMed Central

Chau, K.Y.; Cooper, J.M.; Schapira, A.H.V.

2010-01-01

Rasagiline is a propargylamine and irreversible monoamine oxidase (MAO) B inhibitor used for the treatment of Parkinson's disease (PD). It has demonstrated neuroprotective properties in laboratory studies. Current concepts of PD aetiopathogenesis include the role of alpha-synuclein, protein aggregation, free radical metabolism and mitochondrial dysfunction in contributing to cell death. We have used a combination of alpha-synuclein and free radical mediated toxicity in a dopaminergic cell line to provide a model of nigral toxicity in order to investigate the potential molecular mechanisms that mediate rasagiline protection. We demonstrate that rasagiline protects against cell death induced by the combination of free radicals generated by paraquat and either wild-type or A53T mutant alpha-synuclein over-expression. This protection was associated with a reduction in caspase 3 activation, a reduction in superoxide generation and a trend to ameliorate the fall in mitochondrial membrane potential. Rasagiline induced an increase in cellular glutathione levels. The results support a role for rasagiline in protecting dopaminergic cells against free radical mediated damage and apoptosis in the presence of alpha-synuclein over-expression. The data are of relevance to the interpretation of the potential mechanisms of action of rasagiline in explaining the results of disease modification trials in PD. PMID:20624440
Elevated S100A9 expression in tumor stroma functions as an early recurrence marker for early-stage oral cancer patients through increased tumor cell invasion, angiogenesis, macrophage recruitment and interleukin-6 production

PubMed Central

Fang, Wei-Yu; Chen, Yi-Wen; Hsiao, Jenn-Ren; Liu, Chiang-Shin; Kuo, Yi-Zih; Wang, Yi-Ching; Chang, Kung-Chao; Tsai, Sen-Tien; Chang, Mei-Zhu; Lin, Siao-Han; Wu, Li-Wha

2015-01-01

S100A9 is a calcium-binding protein with two EF-hands and frequently deregulated in several cancer types, however, with no clear role in oral cancer. In this report, the expression of S100A9 in cancer and adjacent tissues from 79 early-stage oral cancer patients was detected by immunohistochemical staining. Although S100A9 protein was present in both tumor and stromal cells, only the early-stage oral cancer patients with high stromal expression had reduced recurrence-free survival. High stromal S100A9 expression was also significantly associated with non-well differentiation and recurrence. In addition to increasing cell migration and invasion, ectopic S100A9 expression in tumor cells promoted xenograft tumorigenesis as well as the dominant expression of myeloid cell markers and pro-inflammatory IL-6. The expression of S100A9 in one stromal component, monocytes, stimulated the aggressiveness of co-cultured oral cancer cells. We also detected the elevation of serum S100A9 levels in early-stage oral cancer patients of a separate cohort of 73 oral cancer patients. The release of S100A9 protein into extracellular milieu enhanced tumor cell invasion, transendothelial monocyte migration and angiogenic activity. S100A9-mediated release of IL-6 requires the crosstalk of tumor cells with monocytes through the activation of NF-κB and STAT-3. Early-stage oral cancer patients with both high S100A9 expression and high CD68+ immune infiltrates in stroma had shortest recurrence-free survival, suggesting the use of both S100A9 and CD68 as poor prognostic markers for oral cancer. Together, both intracellular and extracellular S100A9 exerts a tumor-promoting action through the activation of oral cancer cells and their associated stroma in oral carcinogenesis. PMID:26315114
Elevated S100A9 expression in tumor stroma functions as an early recurrence marker for early-stage oral cancer patients through increased tumor cell invasion, angiogenesis, macrophage recruitment and interleukin-6 production.

PubMed

Fang, Wei-Yu; Chen, Yi-Wen; Hsiao, Jenn-Ren; Liu, Chiang-Shin; Kuo, Yi-Zih; Wang, Yi-Ching; Chang, Kung-Chao; Tsai, Sen-Tien; Chang, Mei-Zhu; Lin, Siao-Han; Wu, Li-Wha

2015-09-29

S100A9 is a calcium-binding protein with two EF-hands and frequently deregulated in several cancer types, however, with no clear role in oral cancer. In this report, the expression of S100A9 in cancer and adjacent tissues from 79 early-stage oral cancer patients was detected by immunohistochemical staining. Although S100A9 protein was present in both tumor and stromal cells, only the early-stage oral cancer patients with high stromal expression had reduced recurrence-free survival. High stromal S100A9 expression was also significantly associated with non-well differentiation and recurrence. In addition to increasing cell migration and invasion, ectopic S100A9 expression in tumor cells promoted xenograft tumorigenesis as well as the dominant expression of myeloid cell markers and pro-inflammatory IL-6. The expression of S100A9 in one stromal component, monocytes, stimulated the aggressiveness of co-cultured oral cancer cells. We also detected the elevation of serum S100A9 levels in early-stage oral cancer patients of a separate cohort of 73 oral cancer patients. The release of S100A9 protein into extracellular milieu enhanced tumor cell invasion, transendothelial monocyte migration and angiogenic activity. S100A9-mediated release of IL-6 requires the crosstalk of tumor cells with monocytes through the activation of NF-κB and STAT-3. Early-stage oral cancer patients with both high S100A9 expression and high CD68+ immune infiltrates in stroma had shortest recurrence-free survival, suggesting the use of both S100A9 and CD68 as poor prognostic markers for oral cancer. Together, both intracellular and extracellular S100A9 exerts a tumor-promoting action through the activation of oral cancer cells and their associated stroma in oral carcinogenesis.
Adventitious viruses in insect cell lines used for recombinant protein expression.

PubMed

Geisler, Christoph; Jarvis, Donald L

2018-04-01

Insect cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several insect cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those cell lines. Furthermore, these findings raised serious concerns about the safety of biologicals produced using those cell lines. In response, new insect cell lines lacking adventitious viruses have been isolated for use as improved research tools and safer biological manufacturing platforms. Here, we review the scientific and patent literature on adventitious viruses found in insect cell lines, affected cell lines, and new virus-free cell lines. Copyright © 2017 Elsevier Inc. All rights reserved.
Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis

PubMed Central

Chen, Liye; Ridley, Anna; Hammitzsch, Ariane; Al-Mossawi, Mohammad Hussein; Bunting, Helen; Georgiadis, Dimitris; Chan, Antoni; Kollnberger, Simon; Bowness, Paul

2016-01-01

Objective Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. Methods Flow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3ε-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition. Results The AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3ε-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion. Conclusions ERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment. PMID:26130142
A model of lytic, latent, and reactivating varicella-zoster virus infections in isolated enteric neurons.

PubMed

Gershon, Anne A; Chen, Jason; Gershon, Michael D

2008-03-01

Because human primary afferent neurons are not readily obtained, we sought to develop a model in which the lytic, latent, and reactivating phases of varicella-zoster virus (VZV) infection were recapitulated in neurons from an animal source. Enteric neurons were obtained from the small intestine of adult guinea pigs and from the bowel of fetal mice. Latency was established when these neurons were infected by cell-free VZV in the absence of fibroblasts or other cells of mesodermal origin. In contrast, lytic infection ensued when fibroblasts were present or when the enteric neurons were infected by cell-associated VZV. Latency was associated with the expression of a limited subset of viral genes, the products of which were restricted to the cytoplasm. Lysis was associated with the expression of viral glycoproteins, nuclear translocation of latency-associated gene products, and rapid cell death. Reactivation was accomplished by expressing VZV open reading frame (ORF) 61p or herpes simplex virus ICP0 in latently infected neurons. Isolated enteric neurons from guinea pigs and mice recapitulate latent gene expression in human cranial nerve and dorsal root ganglia. Expression of latency-associated VZV gene products was detected in 88% of samples of adult human intestine, suggesting that VZV not only infects enteric neurons but also is latent in the human enteric nervous system. This in vitro model should facilitate further understanding of latency and reactivation of VZV.
Modulation of c-fms proto-oncogene in an ovarian carcinoma cell line by a hammerhead ribozyme.

PubMed Central

Yokoyama, Y.; Morishita, S.; Takahashi, Y.; Hashimoto, M.; Tamaya, T.

1997-01-01

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9376277
Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

PubMed

Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

2010-01-01

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.
Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells.

PubMed

Wu, Ting-Hsiang; Sagullo, Enrico; Case, Dana; Zheng, Xin; Li, Yanjing; Hong, Jason S; TeSlaa, Tara; Patananan, Alexander N; McCaffery, J Michael; Niazi, Kayvan; Braas, Daniel; Koehler, Carla M; Graeber, Thomas G; Chiou, Pei-Yu; Teitell, Michael A

2016-05-10

mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells. Copyright © 2016 Elsevier Inc. All rights reserved.
Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics.

PubMed

Valtink, Monika; Gruschwitz, Rita; Funk, Richard H W; Engelmann, Katrin

2008-01-01

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 +/- 14.5 h and that of H9C1 cells 44.05 +/- 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase alpha1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. Copyright 2008 S. Karger AG, Basel.
Development of a dose-controlled multiculture cell exposure chamber for efficient delivery of airborne and engineered nanoparticles

NASA Astrophysics Data System (ADS)

Asimakopoulou, Akrivi; Daskalos, Emmanouil; Lewinski, Nastassja; Riediker, Michael; Papaioannou, Eleni; Konstandopoulos, Athanasios G.

2013-04-01

In order to study the various health influencing parameters related to engineered nanoparticles as well as to soot emitted by Diesel engines, there is an urgent need for appropriate sampling devices and methods for cell exposure studies that simulate the respiratory system and facilitate associated biological and toxicological tests. The objective of the present work was the further advancement of a Multiculture Exposure Chamber (MEC) into a dose-controlled system for efficient delivery of nanoparticles to cells. It was validated with various types of nanoparticles (Diesel engine soot aggregates, engineered nanoparticles for various applications) and with state-of-the-art nanoparticle measurement instrumentation to assess the local deposition of nanoparticles on the cell cultures. The dose of nanoparticles to which cell cultures are being exposed was evaluated in the normal operation of the in vitro cell culture exposure chamber based on measurements of the size specific nanoparticle collection efficiency of a cell free device. The average efficiency in delivering nanoparticles in the MEC was approximately 82%. The nanoparticle deposition was demonstrated by Transmission Electron Microscopy (TEM). Analysis and design of the MEC employs Computational Fluid Dynamics (CFD) and true to geometry representations of nanoparticles with the aim to assess the uniformity of nanoparticle deposition among the culture wells. Final testing of the dose-controlled cell exposure system was performed by exposing A549 lung cell cultures to fluorescently labeled nanoparticles. Delivery of aerosolized nanoparticles was demonstrated by visualization of the nanoparticle fluorescence in the cell cultures following exposure. Also monitored was the potential of the aerosolized nanoparticles to generate reactive oxygen species (ROS) (e.g. free radicals and peroxides generation), thus expressing the oxidative stress of the cells which can cause extensive cellular damage or damage on DNA.

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

PubMed Central

de la Mora, Javier; Uchida, Kaoru; del Campo, Ana Martínez; Camarena, Laura; Aizawa, Shin-Ichi

2015-01-01

ABSTRACT Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 μm and a diameter of 1.4 μm, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (right-handed) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. IMPORTANCE R. sphaeroides possesses two functional sets of flagellar genes. The fla1 genes are normally expressed in the laboratory and were acquired by horizontal transfer. The fla2 genes are endogenous and are expressed in a Fla1− mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism. PMID:26124240
In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse

PubMed Central

McGinty, James; Stuckey, Daniel W.; Soloviev, Vadim Y.; Laine, Romain; Wylezinska-Arridge, Marzena; Wells, Dominic J.; Arridge, Simon R.; French, Paul M. W.; Hajnal, Joseph V.; Sardini, Alessandro

2011-01-01

Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct. PMID:21750768
Altered Hepa1-6 cells by dimethyl sulfoxide (DMSO)-treatment induce anti-tumor immunity in vivo.

PubMed

Jiang, Zhengyu; Zhang, Hongxia; Wang, Ye; Yu, Bin; Wang, Chen; Liu, Changcheng; Lu, Juan; Chen, Fei; Wang, Minjun; Yu, Xinlu; Lin, Jiahao; Pan, Xinghua; Wang, Pin; Zhu, Haiying

2016-02-23

Cancer immunotherapy is the use of the immune system to treat cancer. Our current research proposed an optional strategy of activating immune system involving in cancer immunotherapy. When being treated with 2% DMSO in culture medium, Hepa1-6 cells showed depressed proliferation with no significant apoptosis or decreased viability. D-hep cells, Hepa1-6 cells treated with DMSO for 7 days, could restore to the higher proliferation rate in DMSO-free medium, but alteration of gene expression profile was irreversible. Interestingly, tumors from D-hep cells, not Hepa1-6 cells, regressed in wild-type C57BL/6 mice whereas D-hep cells exhibited similar tumorigenesis as Hep1-6 cells in immunodeficient mice. As expected, additional Hepa1-6 cells failed to form tumors in the D-hep-C57 mice in which D-hep cells were eliminated. Further research confirmed that D-hep-C57 mice established anti-tumor immunity against Hepa1-6 cells. Our research proposed viable tumor cells with altered biological features by DMSO-treatment could induce anti-tumor immunity in vivo.
Involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells.

PubMed

Takahashi, Kaede; Fukushima, Kaori; Onishi, Yuka; Minami, Kanako; Otagaki, Shiho; Ishimoto, Kaichi; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

2018-08-01

Free fatty acid receptor 1 (FFA1) and FFA4 mediate a variety of biological responses through binding of medium- and long-chain free fatty acids. The aim of this study was to investigate an involvement of FFA1 and FFA4 in the regulation of cellular functions during tumor progression in colon cancer cells. The long-term fluorouracil (5-FU) and cisplatin (CDDP) treated cells were generated from DLD1 cells (DLD-5FU and DLD-CDDP cells, respectively). FFAR1 expressions were lower in DLD-5FU and DLD-CDDP cells than in DLD1 cells. In contrast, DLD-5FU and DLD-CDDP cells showed the high FFAR4 expressions, compared with DLD1 cells. The cell motile activities of DLD-5FU and DLD-CDDP cells were reduced by GW9508 which is an agonist of FFA1 and FFA4. Moreover, GW1100, an antagonist of FFA1, inhibited the cell motile activities of DLD-5FU and DLD-CDDP cells. To evaluate whether FFA1 and FFA4 regulate the enhancement of cell motility, invasion and colony formation, highly migratory (hmDLD1) cells were established from DLD1 cells. FFAR1 expression was significantly higher in hmDLD1 cells than in DLD1 cells, but no change of FFAR4 expression was observed. The elevated cell motile and invasive activities and colony formation of hmDLD1 cells were suppressed by FFA1 inhibition. These results suggest that FFA1 and FFA4 are involved in the regulation of cellular functions during tumor progression in colon cancer DLD1 cells. Copyright © 2018 Elsevier Inc. All rights reserved.
High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation[S

PubMed Central

McGrath, Kristine C.; Li, Xiao Hong; Whitworth, Phillippa T.; Kasz, Robert; Tan, Joanne T.; McLennan, Susan V.; Celermajer, David S.; Barter, Philip J.; Rye, Kerry-Anne; Heather, Alison K.

2014-01-01

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation. PMID:24347528
Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

PubMed Central

Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

2015-01-01

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing. PMID:20705106
Targeted Nanocurcumin Therapy Using Annexin A2 Anitbody Improves Tumor Accumulation and Therapeutic Efficacy Against Highly Metastatic Breast Cancer.

PubMed

Mukerjee, Anindita; Ranjan, Anmalendu P; Vishwanatha, Jamboor K

2016-07-01

A major challenge in pharmaceutical research is effective targeting strategies to their sites of action. Emerging knowledge and the current progress in nanotechnology based delivery systems has opened up exciting ways towards successful targeted nanodelivery systems. For cancer therapy, nanoparticle-based drug formulations hold several advantages over free drugs, including improved pharmacokinetics, enhanced tumor accumulation, reduced systemic exposure and side effects and better patient compliance. The goal of this study was to validate the in vivo targeting potential and evaluate the combinatorial therapeutic potential of novel Annexin A2 (AnxA2) antibody-conjugated curcumin loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (AnxA2-CPNP) against metastatic breast cancer. As a first step, we demonstrated that the cell-surface expression of AnxA2 is increases during breast cancer progression with very high expression in highly malignant cancer cells and basal expression in non-malignant cells. This confirmed AnxA2 as an excellent target for targeting our curcumin nanoparticles. Our results indicate that AnxA2-CPNP showed increased uptake in highly metastatic breast cancer cells than untargeted nanoparticles due to the differential AnxA2 expression. Cell viability, plasmin generation and wound healing assays reveal that AnxA2-CPNPs effectively inhibited cell proliferation, invasion and migration, key elements for cancer growth and metastasis. Further, angiogenesis assay illustrated that AnxA2-CPNPs decreased the formation of tube capillaries, thus inhibiting neoangiogenesis, a critical element in tumor growth. Live animal imaging demonstrated that AnxA2-PNPs and AnxA2-CPNPs effectively targeted and accumulated in the tumor as seen by the increased fluorescence intensity on the live scans. Xenograft studies in mice showed significant regression of breast tumor as a result of both effective targeting, accumulation and sustained release of curcumin in the tumor. In conclusion, AnxA2-CPNPs were successfully validated for their breast tumor targeting potential and its improved therapeutic efficacy against metastatic breast cancer.
Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

PubMed

Lee, Kyung-Ho; Kim, Dong-Myung

2013-11-01

Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
IGF-1R Promotes Symmetric Self-Renewal and Migration of Alkaline Phosphatase+ Germ Stem Cells through HIF-2α-OCT4/CXCR4 Loop under Hypoxia.

PubMed

Kuo, Yung-Che; Au, Heng-Kien; Hsu, Jue-Liang; Wang, Hsiao-Feng; Lee, Chiung-Ju; Peng, Syue-Wei; Lai, Ssu-Chuan; Wu, Yu-Chih; Ho, Hong-Nerng; Huang, Yen-Hua

2018-02-13

Hypoxia cooperates with endocrine signaling to maintain the symmetric self-renewal proliferation and migration of embryonic germline stem cells (GSCs). However, the lack of an appropriate in vitro cell model has dramatically hindered the understanding of the mechanism underlying this cooperation. Here, using a serum-free system, we demonstrated that hypoxia significantly induced the GSC mesenchymal transition, increased the expression levels of the pluripotent transcription factor OCT4 and migration-associated proteins (SDF-1, CXCR4, IGF-1, and IGF-1R), and activated the cellular expression and translocalization of the CXCR4-downstream proteins ARP3/pFAK. The underlying mechanism involved significant IGF-1/IGF-1R activation of OCT4/CXCR4 expression through HIF-2α regulation. Picropodophyllin-induced inhibition of IGF-1R phosphorylation significantly suppressed hypoxia-induced SDF-1/CXCR4 expression and cell migration. Furthermore, transactivation between IGF-1R and CXCR4 was involved. In summary, we demonstrated that niche hypoxia synergistically cooperates with its associated IGF-1R signaling to regulate the symmetric division (self-renewal proliferation) and cell migration of alkaline phosphatase-positive GSCs through HIF-2α-OCT4/CXCR4 during embryogenesis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Vaccination with OVA-bound nanoparticles encapsulating IL-7 inhibits the growth of OVA-expressing E.G7 tumor cells in vivo.

PubMed

Toyota, Hiroko; Yanase, Noriko; Yoshimoto, Takayuki; Harada, Mitsunori; Kato, Yasuki; Mizuguchi, Junichiro

2015-01-01

Immunotherapy has gained special attention due to its specific effects on tumor cells and systemic action to block metastasis. We recently demonstrated that ovalbumin (OVA) conjugated to the surface of nanoparticles (NPs) (OVA‑NPs) can manipulate humoral immune responses. In the present study, we aimed to ascertain whether vaccination with OVA-NPs entrapping IL-7 (OVA-NPs-IL-7) are able to induce antitumor immune responses in vivo. Pretreatment with a subcutaneous inoculation of OVA-NPs delayed the growth of thymic lymphoma cells expressing a model tumor antigen OVA (E.G7-OVA), and OVA-NPs-IL-7 substantially blocked the growth of E.G7-OVA tumor cells, although NPs-IL-7 alone had a meager effect, as assessed by the mean tumor size and the percentage of tumor-free mice. However, pretreatment with OVA-NPs-IL-7 failed to reduce the growth of parental thymic tumor cells, suggesting that the antitumor effect was antigen-specific. A tetramer assay revealed that vaccination with OVA-NPs-IL-7 tended to enhance the proportion of cytotoxic T cells (CTLs) specific for OVA. When the tumor-free mice inoculated with OVA-NPs-IL-7 plus EG.7 cells were rechallenged with E.G7-OVA cells, they demonstrated reduced growth compared with that in the control mice. Thus, a single subcutaneous injection of OVA-NPs-IL-7 into mice induced tumor-specific and also memory-like immune responses, resulting in regression of tumor cells. Antigens on NPs entrapping IL-7 would be a promising carrier to develop and enhance immune responses, including humoral and cellular immunity as well as a method of drug delivery to a specific target of interest.
Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane

PubMed Central

Uhlemann, Eva-Maria E; Pierson, Hannah E; Fillingame, Robert H; Dmitriev, Oleg Y

2012-01-01

NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes. PMID:22162071
Enhancing Docetaxel Delivery to Multidrug-Resistant Cancer Cells with Albumin-Coated Nanocrystals.

PubMed

Gad, Sheryhan F; Park, Joonyoung; Park, Ji Eun; Fetih, Gihan N; Tous, Sozan S; Lee, Wooin; Yeo, Yoon

2018-01-29

Intravenous delivery of poorly water-soluble anticancer drugs such as docetaxel (DTX) is challenging due to the low bioavailability and the toxicity related to solubilizing excipients. Colloidal nanoparticles are used as alternative carriers, but low drug loading capacity and circulation instability limit their clinical translation. To address these challenges, DTX nanocrystals (NCs) were prepared using Pluronic F127 as an intermediate stabilizer and albumin as a functional surface modifier, which were previously found to be effective in producing small and stable NCs. We hypothesize that the albumin-coated DTX NCs (DTX-F-alb) will remain stable in serum-containing medium so as to effectively leverage the enhanced permeability and retention effect. In addition, the surface-bound albumin, in its native form, may contribute to cellular transport of NCs through interactions with albumin-binding proteins such as secreted protein acidic and rich in cysteine (SPARC). DTX-F-alb NCs showed sheet-like structure with an average length, width, and thickness of 284 ± 96, 173 ± 56, and 40 ± 8 nm and remained stable in 50% serum solution at a concentration greater than 10 μg/mL. Cytotoxicity and cellular uptake of DTX-F-alb and unformulated (free) DTX were compared on three cell lines with different levels of SPARC expression and DTX sensitivity. While the uptake of free DTX was highly dependent on DTX sensitivity, DTX-F-alb treatment resulted in relatively consistent cellular levels of DTX. Free DTX was more efficient in entering drug-sensitive B16F10 and SKOV-3 cells than DTX-F-alb, with consistent cytotoxic effects. In contrast, multidrug-resistant NCI/ADR-RES cells took up DTX-F-alb more than free DTX with time and responded better to the former. This difference was reduced by SPARC knockdown. The high SPARC expression level of NCI/ADR-RES cells, the known affinity of albumin for SPARC, and the opposing effect of SPARC knockdown support that DTX-F-alb have exploited the surface-bound albumin-SPARC interaction in entering NCI/ADR-RES cells. Albumin-coated NC system is a promising formulation for the delivery of hydrophobic anticancer drugs to multidrug-resistant tumors.
Neuroligin 4X overexpression in human breast cancer is associated with poor relapse-free survival.

PubMed

Henderson, Henry J; Karanam, Balasubramanyam; Samant, Rajeev; Vig, Komal; Singh, Shree R; Yates, Clayton; Bedi, Deepa

2017-01-01

The molecular mechanisms involved in breast cancer progression and metastasis still remain unclear to date. It is a heterogeneous disease featuring several different phenotypes with consistently different biological characteristics. Neuroligins are neural cell adhesion molecules that have been implicated in heterotopic cell adhesion. In humans, alterations in neuroligin genes are implicated in autism and other cognitive diseases. Until recently, neuroligins have been shown to be abundantly expressed in blood vessels and also play a role implicated in the growth of glioma cells. Here we report increased expression of neuroligin 4X (NLGN4X) in breast cancer. We found NLGN4X was abundantly expressed in breast cancer tissues. NLGN4X expression data for all breast cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) was analyzed. Correlation between NLGN4X levels and clinicopathologic parameters were analyzed within Oncomine datasets. Evaluation of these bioinfomatic datasets results revealed that NLGN4X expression was higher in triple negative breast cancer cells, particularly the basal subtype and tissues versus non-triple-negative sets. Its level was also observed to be higher in metastatic tissues. RT-PCR, flow cytometry and immunofluorescence study of MDA-MB-231 and MCF-7 breast cancer cells validated that NLGN4X was increased in MDA-MB-231. Knockdown of NLGN4X expression by siRNA decreased cell proliferation and migration significantly in MDA-MB-231 breast cancer cells. NLGN4X knockdown in MDA-MB-231 cells resulted in induction of apoptosis as determined by annexin staining, elevated caspase 3/7 and cleaved PARP by flow cytometry. High NLGN4X expression highly correlated with decrease in relapse free-survival in TNBC. NLGN4X might represent novel biomarkers and therapeutic targets for breast cancer. Inhibition of NLGN4X may be a new target for the prevention and treatment of breast cancer.
Changes in proto-oncogene expression associated with reversal of macrophage-like differentiation of HL 60 cells.

PubMed

Studzinski, G P; Brelvi, Z S

1987-07-01

Prolonged exposure to 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] of 2 sublines (AB-2 and AB-26) of human promyelocytic HL 60 leukemia cells produced increased adherence of the cells to the culture substratum. Advantage was taken of this property to separate physically a population of cells highly enriched in macrophage-like forms. When these differentiated cells were placed in culture medium free of 1,25(OH)2D3, there was a rapid reversal of the features of the differentiated phenotype, monitored by the loss of alpha-naphthyl butyrate esterase activity and the loss of adherence to the substrate. The reversal was accompanied by the resumption of normal rates of DNA synthesis, mitosis, and reaccumulation of c-myc and c-myb transcripts. The levels of transcripts of oncogenes c-fos and c-fms, which became abundant in the phenotypically differentiated cultures, declined along with the loss of adhesiveness and reversion to more primitive myeloblastic forms. These changes in proto-oncogene expression became evident before cell proliferation resumed, thereby excluding the diluting effect of the outgrowth of undifferentiated cells. It is concluded that in this system there is no firm commitment to terminal, as opposed to early, differentiation in the great majority of the cells and that the expression of the monocytic maturation-associated genes c-fos and c-fms is down-regulated when macrophage-like cells dedifferentiate. This strengthens the case for an association between macrophage differentiation and the expression of oncogenes c-fos and c-fms.
Binary colloidal crystals (BCCs) as a feeder-free system to generate human induced pluripotent stem cells (hiPSCs)

PubMed Central

Wang, Peng-Yuan; Hung, Sandy Shen-Chi; Thissen, Helmut; Kingshott, Peter; Wong, Raymond Ching-Bong

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of differentiating into any cell type and provide significant advances to cell therapy and regenerative medicine. However, the current protocol for hiPSC generation is relatively inefficient and often results in many partially reprogrammed colonies, which increases the cost and reduces the applicability of hiPSCs. Biophysical stimulation, in particular from tuning cell-surface interactions, can trigger specific cellular responses that could in turn promote the reprogramming process. In this study, human fibroblasts were reprogrammed into hiPSCs using a feeder-free system and episomal vectors using novel substrates based on binary colloidal crystals (BCCs). BCCs are made from two different spherical particle materials (Si and PMMA) ranging in size from nanometers to micrometers that self-assemble into hexagonal close-packed arrays. Our results show that the BCCs, particularly those made from a crystal of 2 μm Si and 0.11 μm PMMA particles (2SiPM) facilitate the reprogramming process and increase the proportion of fully reprogrammed hiPSC colonies, even without a vitronectin coating. Subsequent isolation of clonal hiPSC lines demonstrates that they express pluripotent markers (OCT4 and TRA-1-60). This proof-of-concept study demonstrates that cell reprogramming can be improved on substrates where surface properties are tailored to the application. PMID:27833126
Simvastatin prevents triple-negative breast cancer metastasis in pre-clinical models through regulation of FOXO3a

PubMed Central

Wolfe, Adam R.; Debeb, Bisrat G.; Lacerda, Lara; Larson, Richard; Bambhroliya, Arvind; Huang, Xuelin; Bertucci, Francois; Finetti, Pascal; Birnbaum, Daniel; Van Laere, Steven; Diagaradjan, Parmeswaran; Ruffell, Brian; Trenton, Nicholaus J.; Chu, Khoi; Hittelman, Walter; Diehl, Michael; Levental, Ilya; Ueno, Naoto T.; Woodward, Wendy A.

2016-01-01

Purpose We previously reported using statins was correlated with improved metastasis free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple negative breast cancer (TNBC) cells. Experimental Design TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in 8 public breast cancer gene expression data sets including 1,479 patients. Results Simvastatin increased G1/S phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pretreatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Conclusions Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted. PMID:26590814
Simvastatin prevents triple-negative breast cancer metastasis in pre-clinical models through regulation of FOXO3a.

PubMed

Wolfe, Adam R; Debeb, Bisrat G; Lacerda, Lara; Larson, Richard; Bambhroliya, Arvind; Huang, Xuelin; Bertucci, Francois; Finetti, Pascal; Birnbaum, Daniel; Van Laere, Steven; Diagaradjan, Parmeswaran; Ruffell, Brian; Trenton, Nicholaus J; Chu, Khoi; Hittelman, Walter; Diehl, Michael; Levental, Ilya; Ueno, Naoto T; Woodward, Wendy A

2015-12-01

We previously reported using statins was correlated with improved metastasis-free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple-negative breast cancer (TNBC) cells. TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in eight public breast cancer gene expression datasets including 1479 patients. Simvastatin increased G1/S-phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pre-treatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.
ARHGAP42 promotes cell migration and invasion involving PI3K/Akt signaling pathway in nasopharyngeal carcinoma.

PubMed

Hu, Qian; Lin, Xiao; Ding, Linxiaoxiao; Zeng, Yinduo; Pang, Danmei; Ouyang, Nengtai; Xiang, Yanqun; Yao, Herui

2018-06-24

Rho GTPase-activating protein 42 was identified as an inhibitor of RhoA to maintain normal blood pressure homeostasis. However, the effect of ARHGAP42 in promoting cell malignancy in nasopharyngeal carcinoma is demonstrated in this study. Microarray and real-time quantitative PCR were used for a mRNA profiling of ARHGAP42 in nasopharyngeal primary and metastatic carcinoma tissues. Western blot and immunohistochemical staining were used for detecting the expression of ARHGAP42 protein in nasopharyngeal carcinoma tissues and cell lines. The overexpression and silence experiments of ARHGAP42 were performed in NPC cell lines using siRNA and expressive plasmid for evaluating cancer cell migration and invasion in vitro. Real-time quantitative PCR, western blot, and transwell test were employed for with the function of ARHGAP42 and its antisense lncRNA uc010rul. We confirmed the elevated expression of ARHGAP42 in metastatic NPC tissues of mRNA and protein for the first time. Immunohistochemical analysis indicated that NPC patients with highly ARHGAP42 expression were significantly associated with shorter metastasis-free survival. Knockdown of ARHGAP42 resulted in significant inhibition of nasopharyngeal cancer cell migration and invasion in vitro, and the overexpression of ARHGAP42 showed the opposite effects. In addition, the silence of uc010rul resulted in ARHGAP42 expression decrease and significant inhibition of nasopharyngeal cancer cell migration and invasion. High expression of ARHGAP42 is associated with poor metastasis-free survival of nasopharyngeal carcinoma patients. ARHGAP42 promotes migration and invasion of nasopharyngeal carcinoma cells in vitro; the antisense lncRNA may be involved in this effect. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
Doxorubicin-conjugated bacteriophages carrying anti-MHC class I chain-related A for targeted cancer therapy in vitro.

PubMed

Phumyen, Achara; Jantasorn, Siriporn; Jumnainsong, Amonrat; Leelayuwat, Chanvit

2014-01-01

Cancer therapy by systemic administration of anticancer drugs, besides the effectiveness shown on cancer cells, demonstrated the side effects and cytotoxicity on normal cells. The targeted drug-carrying nanoparticles may decrease the required drug concentration at the site and the distribution of drugs to normal tissues. Overexpression of major histocompatibility complex class I chain-related A (MICA) in cancer is useful as a targeted molecule for the delivery of doxorubicin to MICA-expressing cell lines. The application of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) chemistry was employed to conjugate the major coat protein of bacteriophages carrying anti-MICA and doxorubicin in a mildly acid condition. Doxorubicin (Dox) on phages was determined by double fluorescence of phage particles stained by M13-fluorescein isothiocyanate (FITC) and drug autofluorescence by flow cytometry. The ability of anti-MICA on phages to bind MICA after doxorubicin conjugation was evaluated by indirect enzyme-linked immunosorbent assay. One cervical cancer and four cholangiocarcinoma cell lines expressing MICA were used as models to evaluate targeting activity by cell cytotoxicity test. Flow cytometry and indirect enzyme-linked immunosorbent assay demonstrated that most of the phages (82%) could be conjugated with doxorubicin, and the Dox-carrying phage-displaying anti-MICA (Dox-phage) remained the binding activity against MICA. Dox-phage was more efficient than free drugs in killing all the cell lines tested. The half maximal inhibitory concentration (IC50) values of Dox-phage were lower than those of free drugs at approximately 1.6-6 times depending on MICA expressions and the cell lines tested. Evidently, the application of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide chemistry is effective to conjugate doxorubicin and major coat protein of bacteriophages without destroying binding activity of MICA antibodies. Dox-carrying bacteriophages targeting MICA have been successfully developed and may enable a broad range of applications in cancer-targeting chemotherapy.
Bioengineered intestinal muscularis complexes with long-term spontaneous and periodic contractions

PubMed Central

Wang, Qianqian; Wang, Ke; Solorzano-Vargas, R. Sergio; Lin, Po-Yu; Walthers, Christopher M.; Thomas, Anne-Laure; Martín, Martín G.

2018-01-01

Although critical for studies of gut motility and intestinal regeneration, the in vitro culture of intestinal muscularis with peristaltic function remains a significant challenge. Periodic contractions of intestinal muscularis result from the coordinated activity of smooth muscle cells (SMC), the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). Reproducing this activity requires the preservation of all these cells in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal muscularis cells for months. In this system, SMC expressed the mature marker myosin heavy chain, and multipolar/dipolar ICC, uniaxonal/multipolar neurons and glial cells were present. Furthermore, drugs affecting neural signals, ICC or SMC altered the contractions. Combining this method with scaffolds, contracting cell sheets were formed with organized architecture. With the addition of intestinal epithelial cells, this platform enabled up to 11 types of cells from mucosa, muscularis and serosa to coexist and epithelial cells were stretched by the contracting muscularis cells. The method constitutes a powerful tool for mechanistic studies of gut motility disorders and the functional regeneration of the engineered intestine. PMID:29718926

Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.

PubMed

Piao, Yulan; Hung, Sandy Shen-Chi; Lim, Shiang Y; Wong, Raymond Ching-Bong; Ko, Minoru S H

2014-07-01

Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes. ©AlphaMed Press.
G protein-coupled estrogen receptor 1-mediated effects in the rat myometrium.

PubMed

Tica, Andrei A; Dun, Erica C; Tica, Oana S; Gao, Xin; Arterburn, Jeffrey B; Brailoiu, G Cristina; Oprea, Tudor I; Brailoiu, Eugen

2011-11-01

G protein-coupled estrogen receptor 1 (GPER), also named GPR30, has been previously identified in the female reproductive system. In this study, GPER expression was found in the female rat myometrium by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Using GPER-selective ligands, we assessed the effects of the GPER activation on resting membrane potential and cytosolic Ca(2+) concentration ([Ca(2+)](i)) in rat myometrial cells, as well as on contractility of rat uterine strips. G-1, a specific GPER agonist, induced a concentration-dependent depolarization and increase in [Ca(2+)](i) in myometrial cells. The depolarization was abolished in Na(+)-free saline. G-1-induced [Ca(2+)](i) increase was markedly decreased by nifedipine, a L-type Ca(2+) channel blocker, by Ca(2+)-free or Na(+)-free saline. Intracellular administration of G-1 produced a faster and transitory increase in [Ca(2+)](i), with a higher amplitude than that induced by extracellular application, supporting an intracellular localization of the functional GPER in myometrial cells. Depletion of internal Ca(2+) stores with thapsigargin produced a robust store-activated Ca(2+) entry; the Ca(2+) response to G-1 was similar to the constitutive Ca(2+) entry and did not seem to involve store-operated Ca(2+) entry. In rat uterine strips, administration of G-1 increased the frequency and amplitude of contractions and the area under the contractility curve. The effects of G-1 on membrane potential, [Ca(2+)](i), and uterine contractility were prevented by pretreatment with G-15, a GPER antagonist, further supporting the involvement of GPER in these responses. Taken together, our results indicate that GPER is expressed and functional in rat myometrium. GPER activation produces depolarization, elevates [Ca(2+)](i) and increases contractility in myometrial cells.
An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes.

PubMed

Li, Jinqing; Ichikawa, Tomonaga; Jin, Yu; Hofseth, Lorne J; Nagarkatti, Prakash; Nagarkatti, Mitzi; Windust, Anthony; Cui, Taixing

2010-07-20

Ginseng has been used as a folk medicine for thousands of years in Asia, and has become a popular herbal medicine world-wide. Recent studies have revealed that ginseng, including American ginseng, exerts antioxidant effects in the cardiovascular system; however, the underlying mechanisms are not fully understood. Thus, we investigated role of Nrf2, a master transcription factor of endogenous anti-oxidative defense systems, in the regulation of American ginseng-mediated anti-oxidative actions in cardiomyocytes. A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. H9C2 cells, a rat cardiomyocyte cell line, were exposed to angiotensin II (Ang II) or tumor necrosis factor alpha (TNFalpha) to induce oxidative stress that was examined by measuring formation of reactive oxygen and nitrogen species. Oxidative stress-induced cell death was induced by exogenous addition of hydrogen peroxide (H(2)O(2)). Proteins were measured by Western blot and mRNA expression was determined by quantitative real time PCR. Nrf2-driven transcriptional activity was assessed by antioxidant response element (ARE)-luciferase reporter assay. Direct Nrf2 binding to its target gene promoters was determined by chromatin immunoprecipitation assay. Adenoviral over-expression of Nrf2 shRNA was utilized to knock down Nrf2 in H9C2 cells. Immunochemical staining was applied for Nrf2 expression in the heart. American ginseng induced dramatic increases in Nrf2 protein expression, Nrf2 nuclear translocation, Nrf2 transcriptional activity, direct Nrf2 binding to its target gene promoters, and expression of a group of anti-oxidative genes driven by Nrf2 in H9C2 cells. In addition, American ginseng inhibited Ang II- or TNFalpha-induced free radical formation and H(2)O(2)-induced cell death in H9C2 cells over-expressed with control shRNA but not in the cells over-expressed with Nrf2 shRNA. Finally, oral administration of American ginseng markedly increased Nrf2 activity in murine hearts. These results demonstrate that American ginseng suppresses oxidative stress and oxidative stress-induced cell death in cardiomyocytes through activating the Nrf2 pathway, thereby providing cardioprotection against pathological cardiac remodeling. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.
Cell model and elastic moduli of disordered solids - Low temperature limit

NASA Technical Reports Server (NTRS)

Peng, S. T. J.; Landel, R. F.; Moacanin, J.; Simha, Robert; Papazoglou, Elisabeth

1987-01-01

The cell theory has been previously employed to compute the equation of state of a disordered condensed system. It is now generalized to include anisotropic stresses. The condition of affine deformation is adopted, transforming an orginally spherical into an ellipsoidal cell. With a Lennard-Jones n-m potential between nonbonded centers, the formal expression for the deformational free energy is derived. It is to be evaluated in the limit of the linear elastic range. Since the bulk modulus in this limit is already known, it is convenient to consider a uniaxial deformation. To begin with, restrictions are made to the low-temperature limit in the absence of entropy contributions. Young's modulus and Poisson's ratio then follow.
cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites.

PubMed

Xu, Shuting; Ducroux, Aurélie; Ponnurangam, Aparna; Vieyres, Gabrielle; Franz, Sergej; Müsken, Mathias; Zillinger, Thomas; Malassa, Angelina; Ewald, Ellen; Hornung, Veit; Barchet, Winfried; Häussler, Susanne; Pietschmann, Thomas; Goffinet, Christine

2016-10-12

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection. Copyright © 2016 Elsevier Inc. All rights reserved.
Sperm-Hybrid Micromotor for Targeted Drug Delivery.

PubMed

Xu, Haifeng; Medina-Sánchez, Mariana; Magdanz, Veronika; Schwarz, Lukas; Hebenstreit, Franziska; Schmidt, Oliver G

2018-01-23

A sperm-driven micromotor is presented as a targeted drug delivery system, which is appealing to potentially treat diseases in the female reproductive tract. This system is demonstrated to be an efficient drug delivery vehicle by first loading a motile sperm cell with an anticancer drug (doxorubicin hydrochloride), guiding it magnetically, to an in vitro cultured tumor spheroid, and finally freeing the sperm cell to deliver the drug locally. The sperm release mechanism is designed to liberate the sperm when the biohybrid micromotor hits the tumor walls, allowing it to swim into the tumor and deliver the drug through the sperm-cancer cell membrane fusion. In our experiments, the sperm cells exhibited a high drug encapsulation capability and drug carrying stability, conveniently minimizing toxic side effects and unwanted drug accumulation in healthy tissues. Overall, sperm cells are excellent candidates to operate in physiological environments, as they neither express pathogenic proteins nor proliferate to form undesirable colonies, unlike other cells or microorganisms. This sperm-hybrid micromotor is a biocompatible platform with potential application in gynecological healthcare, treating or detecting cancer or other diseases in the female reproductive system.
Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

PubMed

Gupta, Sanjeev K; Shukla, Pratyoosh

2016-12-01

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.
Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway*

PubMed Central

Henrich, Erik; Ma, Yi; Engels, Ina; Münch, Daniela; Otten, Christian; Schneider, Tanja; Henrichfreise, Beate; Sahl, Hans-Georg; Dötsch, Volker; Bernhard, Frank

2016-01-01

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin. PMID:26620564
Nucleolar protein PES1 is a marker of neuroblastoma outcome and is associated with neuroblastoma differentiation

PubMed Central

Nakaguro, Masato; Kiyonari, Shinichi; Kishida, Satoshi; Cao, Dongliang; Murakami-Tonami, Yuko; Ichikawa, Hitoshi; Takeuchi, Ichiro; Nakamura, Shigeo; Kadomatsu, Kenji

2015-01-01

Neuroblastoma (NB) is a childhood malignant tumor that arises from precursor cells of the sympathetic nervous system. Spontaneous regression is a phenomenon unique to NBs and is caused by differentiation of tumor cells. PES1 is a multifunctional protein with roles in both neural development and ribosome biogenesis. Various kinds of models have revealed the significance of PES1 in neurodevelopment. However, the roles of PES1 in NB tumorigenesis and differentiation have remained unknown. Here we show that NB cases with MYCN amplification and clinically unfavorable stage (INSS stage 4) express higher levels of PES1. High PES1 expression was associated with worse overall and relapse-free survival. In NB cell lines, PES1 knockdown suppressed tumor cell growth and induced apoptosis. This growth inhibition was associated with the expression of NB differentiation markers. However, when the differentiation of NB cell lines was induced by the use of all-trans retinoic acid, there was a corresponding decrease in PES1 expression. Pes1 expression of tumorspheres originated from MYCN transgenic mice also diminished after the induction of differentiation with growth factors. We also reanalyzed the distribution of PES1 in the nucleolus. PES1 was localized in the dense fibrillar component, but not in the granular component of nucleoli. After treatment with the DNA-damaging agent camptothecin, this distribution was dramatically changed to diffuse nucleoplasmic. These data suggest that PES1 is a marker of NB outcome, that it regulates NB cell proliferation, and is associated with NB differentiation. PMID:25557119
Clinicopathological significance of chemokine receptor (CCR1, CCR3, CCR4, CCR5, CCR7 and CXCR4) expression in head and neck squamous cell carcinomas.

PubMed

González-Arriagada, Wilfredo A; Lozano-Burgos, Carlos; Zúñiga-Moreta, Rodrigo; González-Díaz, Paulina; Coletta, Ricardo D

2018-05-24

Head and neck squamous cell carcinoma shows high prevalence of lymph node metastasis at diagnosis, and despite the advances in treatment, the overall 5-year survival is still under 50%. Chemokine receptors have a role in the development and progression of cancer, but their effect in head and neck carcinoma remains poorly characterised. This study aimed to assess the prognostic value of CCR1, CCR3, CCR4, CCR5, CCR7 and CXCR4 in head and neck squamous cell carcinomas. Immunohistochemical expression of chemokine receptors was evaluated in a retrospective cohort of 76 cases of head and neck squamous cell carcinoma. Clinicopathological associations were analysed using the chi-square test, survival curves were analysed according to the Kaplan-Meier method, and the Cox proportional hazard model was applied for multivariate survival analysis. The chemokine receptors were highly expressed in primary carcinomas, except for CCR1 and CCR3. Significant associations were detected, including the associations between CCR5 expression and lymph node metastasis (N stage, P = .03), advanced clinical stage (P = .003), poor differentiation of tumours (P = .05) and recurrence (P = .01). The high expression of CCR5 was also associated with shortened disease-free survival (HR: 2.85, 95% CI: 1.09-8.14, P = .05), but the association did not withstand the Cox multivariate survival analysis. At univariate analysis, high expression of CCR7 was associated with disease-free survival and low levels of CXCR4 were significantly associated with both disease-specific and disease-free survival. These findings show that chemokine receptors may have an important role in head and neck squamous cell carcinoma progression, regional lymph node metastasis and recurrence. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

PubMed

Dalton, George D; Dewey, William L

2006-02-01

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous system and the rest of the body.
Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

PubMed

Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

2017-05-20

Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.
Cinnamic acid shortens the period of the circadian clock in mice.

PubMed

Oishi, Katsutaka; Yamamoto, Saori; Oike, Hideaki; Ohkura, Naoki; Taniguchi, Masahiko

2017-03-01

Cinnamic acid (CA) derivatives have recently received focus due to their anticancer, antioxidant, and antidiabetic properties. The present study aimed to determine the effects of cinnamic acid on the circadian clock, which is a cell-autonomous endogenous system that generates circadian rhythms that govern the behavior and physiology of most organisms. Cinnamic acid significantly shortened the circadian period of PER2::LUC expression in neuronal cells that differentiated from neuronal progenitor cells derived from PER2::LUC mouse embryos. Cinnamic acid did not induce the transient mRNA expression of clock genes such as Per1 and Per2 in neuronal cells, but significantly shortened the half-life of PER2::LUC protein in neuronal cells incubated with actinomycin D, suggested that CA post-transcriptionally affects the molecular clock by decreasing Per2 mRNA stability. A continuous infusion of CA into mice via an Alzet osmotic pump under constant darkness significantly shortened the free-running period of wheel-running rhythms. These findings suggest that CA shortens the circadian period of the molecular clock in mammals.
Vitamin E and mast cells.

PubMed

Zingg, Jean-Marc

2007-01-01

Mast cells play an important role in the immune system by interacting with B and T cells and by releasing several mediators involved in activating other cells. Hyperreactivity of mast cells and their uncontrolled accumulation in tissues lead to increased release of inflammatory mediators contributing to the pathogenesis of several diseases such as rheumatoid arthritis, atherosclerosis, multiple sclerosis, and allergic disorders such as asthma and allergic rhinitis. Interference with mast cell proliferation, survival, degranulation, and migration by synthetic or natural compounds may represent a preventive strategy for the management of these diseases. Natural vitamin E covers a group of eight analogues-the alpha-, beta-, gamma-, and delta-tocopherols and the alpha-, beta-, gamma-, and delta-tocotrienols, but only alpha-tocopherol is efficiently retained by the liver and distributed to peripheral tissues. Mast cells preferentially locate in the proximity of tissues that interface with the external environment (the epithelial surface of the skin, the gastrointestinal mucosa, and the respiratory system), what may render them accessible to treatments with inefficiently retained natural vitamin E analogues and synthetic derivatives. In addition to scavenging free radicals, the natural vitamin E analogues differently modulate signal transduction and gene expression in several cell lines; in mast cells, protein kinase C, protein phosphatase 2A, and protein kinase B are affected by vitamin E, leading to the modulation of proliferation, apoptosis, secretion, and migration. In this chapter, the possibility that vitamin E can prevent diseases with mast cells involvement by modulating signal transduction and gene expression is evaluated.
The role of topotecan for extending the platinum-free interval in recurrent ovarian cancer: an in vitro model.

PubMed

Horowitz, Neil S; Hua, Jun; Gibb, Randall K; Mutch, David G; Herzog, Thomas J

2004-07-01

Topotecan, a novel topoisomerase-I inhibitor, is an active agent of second-line chemotherapy for extending the platinum-free interval (PFI) and improving the chances of a response to platinum in recurrent ovarian cancer patients. The aim of this study was to understand the molecular mechanism of topotecan-based second-line chemotherapy through an in vitro cell culture model and to gain clinical insight into sequencing issues for second-line treatment with novel agents versus retreatment with platinum. The human ovarian cancer cell line A2780 and the cisplatin resistance cell line A2780-CR were separately seeded in 6-well cell culture plates and then exposed to multiple concentrations of cisplatin plus paclitaxel or topotecan for 7 days. Surviving cells were recovered and cultured in drug-free media for 3 weeks and then replated in a 96-well microtiter plate. The LD(50) for these cells was determined by a cytotoxic MTT assay after exposure to multiple clinically relevant concentrations of cisplatin or topotecan. Surviving cells were cultured in drug-free media for an additional 4 weeks at which time the LD(50) was reassessed for each cell population by a second MTT assay. Using RT-PCR and Northern blot hybridization to measure mRNA expression, the molecular profile of these cells in terms of resistance was evaluated for the multidrug-resistant gene (MDR-1), multidrug-resistant protein (MRP), Topoisomerase-I, and beta-Actin. The LD(50) to cisplatin was unchanged in A2780-CR cells treated by topotecan. Those A2780-CR cells originally exposed to higher concentrations of cisplatin became more resistant to cisplatin in the MTT assays, while those A2780-CR cell lines treated with a combination of lower cisplatin concentrations and paclitaxel became more sensitive to cisplatin in the MTT assay (P < 0.01). The second MTT assay demonstrated that the LD(50) for cisplatin in every cell line decreased significantly after a 4-week drug-free interval (P < 0.01). There was no difference in the mRNA expression for MRP or topoisomerase-I regardless of cell line, or type or concentration of chemotherapeutic exposure. The mRNA for MDR-1 was uniquely overexpressed in the cisplatin-resistant cell line A2780-CR9 initially treated with low doses of cisplatin and paclitaxel, but was not amplified in A2780 (P < 0.01). The acquired resistance to cisplatin in A2780 is potentially due to P-glycoprotein-mediated multidrug resistance. This acquired resistance to cisplatin is an unstable phenotype in that some cell populations become sensitive after a drug-free interval and topotecan treatment. This reversal of resistance, however, does not appear to be simply due to loss of MDR-1 expression. While in vivo confirmation is required, agents with novel mechanisms of action offer a strategy to extend the platinum-free interval and thereby improve survival in patients with recurrent ovarian cancer.
Effects of whole body (56)Fe radiation on contextual freezing and Arc-positive cells in the dentate gyrus.

PubMed

Raber, Jacob; Allen, Antiño R; Rosi, Susanna; Sharma, Sourabh; Dayger, Catherine; Davis, Matthew J; Fike, John R

2013-06-01

The space radiation environment contains high-energy charged particles such as (56)Fe, which could pose a significant hazard to hippocampal function in astronauts during and after the mission(s). The mechanisms underlying impairments in cognition are not clear but might involve alterations in the percentage of neurons in the dentate gyrus expressing the plasticity-related immediate early gene Arc. Previously, we showed effects of cranial (56)Fe irradiation on hippocampus-dependent contextual freezing and on the percentage of Arc-positive cells in the enclosed, but not free, blade. Because it is unclear whether whole body (56)Fe irradiation causes similar effects on these markers of hippocampal function, in the present study we quantified the effects of whole body (56)Fe irradiation (600MeV, 0.5 or 1Gy) on hippocampus-dependent and hippocampus-independent cognitive performance and determined whether these effects were associated with changes in Arc expression in the enclosed and free blades of the dentate gyrus. Whole body (56)Fe irradiation impacted contextual but not cued fear freezing and the percentage of Arc-positive cells in the enclosed and free blades. In mice tested for contextual freezing, there was a correlation between Arc-positive cells in the enclosed and free blades. In addition, in mice irradiated with 0.5Gy, contextual freezing in the absence of aversive stimuli correlated with the percentage of Arc-positive cells in the enclosed blade. In mice tested for cued freezing, there was no correlation between Arc-positive cells in the enclosed and free blades. In contrast, cued freezing in the presence or absence of aversive stimuli correlated with Arc-positive cells in the free blade. In addition, in mice irradiated with 1Gy cued freezing in the absence of aversive stimuli correlated with the percentage of Arc-positive neurons in the free blade. These data indicate that while whole body (56)Fe radiation affects contextual freezing and Arc-positive cells in the dentate gyrus, the enclosed blade might be more important for contextual freezing while the free blade might be more important for cued freezing. Copyright © 2013 Elsevier B.V. All rights reserved.
Insights to the effects of free cells on community structure of attached cells and chalcopyrite bioleaching during different stages.

PubMed

Feng, Shoushuai; Yang, Hailin; Wang, Wu

2016-01-01

The effects of free cells on community structure of attached cells and chalcopyrite bioleaching by Acidithiobacillus sp. during different stages were investigated. The attached cells of Acidithiobacillus thiooxidans owned the community advantage from 14thd to the end of bioprocess in the normal system. The community structure of attached cells was greatly influenced in the free cells-deficient systems. Compared to A. thiooxidans, the attached cells community of Acidithiobacillus ferrooxidans had a higher dependence on its free cells. Meanwhile, the analysis of key biochemical parameters revealed that the effects of free cells on chalcopyrite bioleaching in different stages were diverse, ranging from 32.8% to 64.3%. The bioleaching contribution of free cells of A. ferrooxidans in the stationary stage (8-14thd) was higher than those of A. thiooxidans, while the situation was gradually reversed in the jarosite passivation inhibited stage (26-40thd). These results may be useful in guiding chalcopyrite bioleaching. Copyright © 2015 Elsevier Ltd. All rights reserved.
Predictive value of Sox2 expression in transurethral resection specimens in patients with T1 bladder cancer.

PubMed

Ruan, Jun; Wei, Bingbing; Xu, Zhuoqun; Yang, Shudong; Zhou, You; Yu, Minhong; Liang, Jiabei; Jin, Ke; Huang, Xing; Lu, Peng; Cheng, Huan

2013-03-01

Sox2 is thought to be an important regulator of self-renewal in embryonic stem cell. According to the cancer stem cell (CSC) theory, the overexpression of Sox2 is potentially involved in carcinogenesis and could affect tumor recurrence and metastasis. Previous study proved Sox2 might be prognostic marker for multiple human malignancies. The purpose of this study was to investigate the clinicopathological significance of Sox2 expression in human non-muscle-invasive bladder cancer. We examined Sox2 expression in 32 paired non-muscle-invasive bladder cancer tissues and adjacent non-cancerous tissues by quantitative real-time RT-PCR (qrtRT-PCR). In addition, we analyzed Sox2 and Ki-67 expression in 126 non-muscle-invasive bladder cancer samples and bladder cancer cell line T24 by immunohistochemistry and immunofluorescence assays. The recurrence-free survival was determined by Kaplan-Meier method and log-rank test. Cox regression was adopted for univariate and multivariate analyses of prognostic factors. The expression of Sox2 was significantly increased in non-muscle-invasive bladder cancer tissues. Sox2 expression was significantly correlated with that of Ki-67 (P < 0.001). The expression of Sox2 was significantly associated with tumor size (P = 0.006), tumor number (P = 0.037), and tumor grade (P < 0.001). Patients with high Sox2 expression had significantly poorer recurrence-free survival (P = 0.0002) when compared with patients with the low expression of Sox2. On multivariate analysis, Sox2 expression and tumor grade were found to be independent prognostic factors for recurrence-free survival (P < 0.05). Our data suggested for the first time that the high expression of Sox2 may contribute to the development of non-muscle-invasive bladder cancer and serve as a novel prognostic marker in patients with T1 bladder cancer.
Microfluorimetric analysis of a purinergic receptor (P2X7) in GH4C1 rat pituitary cells: effects of a bioactive substance produced by Pfiesteria piscicida.

PubMed Central

Melo, A C; Moeller, P D; Glasgow, H; Burkholder, J M; Ramsdell, J S

2001-01-01

Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP. PMID:11677182
Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents.

PubMed

Liu, Yanfeng; Wang, Ying; Gao, Yongxing; Forbes, Jessica A; Qayyum, Rehan; Becker, Lewis; Cheng, Linzhao; Wang, Zack Z

2015-04-01

Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animal-derived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administration-approved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). We used this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34(+)CD45(+) hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41(+)CD42a(+) MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41(+)CD42a(+) MKs that also expressed the MK markers CD42b and CD61 and displayed polyploidy (≥16% of derived cells with DNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo. ©AlphaMed Press.

Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells

PubMed Central

Smith, Stephen P.; Scarpini, Cinzia G.; Groves, Ian J.; Odle, Richard I.; Coleman, Nicholas

2016-01-01

Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of ‘master regulators’ for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins. PMID:27457222
Nodal Lymphangiogenesis and Metastasis

PubMed Central

Hirakawa, Satoshi; Detmar, Michael; Kerjaschki, Dontscho; Nagamatsu, Shogo; Matsuo, Keitaro; Tanemura, Atsushi; Kamata, Nobuyuki; Higashikawa, Koichiro; Okazaki, Hidenori; Kameda, Kenji; Nishida-Fukuda, Hisayo; Mori, Hideki; Hanakawa, Yasushi; Sayama, Koji; Shirakata, Yuji; Tohyama, Mikiko; Tokumaru, Sho; Katayama, Ichiro; Hashimoto, Koji

2009-01-01

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget’s disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-β1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis. PMID:19815713
Production of biopharmaceuticals and vaccines in plants via the chloroplast genome.

PubMed

Daniell, Henry

2006-10-01

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.
In vitro expansion and differentiation of rat pancreatic duct-derived stem cells into insulin secreting cells using a dynamicthree-dimensional cell culture system.

PubMed

Chen, X C; Liu, H; Li, H; Cheng, Y; Yang, L; Liu, Y F

2016-06-27

In this study, a dynamic three-dimensional cell culture technology was used to expand and differentiate rat pancreatic duct-derived stem cells (PDSCs) into islet-like cell clusters that can secrete insulin. PDSCs were isolated from rat pancreatic tissues by in situ collagenase digestion and density gradient centrifugation. Using a dynamic three-dimensional culture technique, the cells were expanded and differentiated into functional islet-like cell clusters, which were characterized by morphological and phenotype analyses. After maintaining 1 x 108 isolated rat PDSCs in a dynamic three-dimensional cell culture for 7 days, 1.5 x 109 cells could be harvested. Passaged PDSCs expressed markers of pancreatic endocrine progenitors, including CD29 (86.17%), CD73 (90.73%), CD90 (84.13%), CD105 (78.28%), and Pdx-1. Following 14 additional days of culture in serum-free medium with nicotinamide, keratinocyte growth factor (KGF), and b fibroblast growth factor (FGF), the cells were differentiated into islet-like cell clusters (ICCs). The ICC morphology reflected that of fused cell clusters. During the late stage of differentiation, representative clusters were non-adherent and expressed insulin indicated by dithizone (DTZ)-positive staining. Insulin was detected in the extracellular fluid and cytoplasm of ICCs after 14 days of differentiation. Additionally, insulin levels were significantly higher at this time compared with the levels exhibited by PDSCs before differentiation (P < 0.01). By using a dynamic three-dimensional cell culture system, PDSCs can be expanded in vitro and can differentiate into functional islet-like cell clusters.
Biological adhesion of the flatworm Macrostomum lignano relies on a duo-gland system and is mediated by a cell type-specific intermediate filament protein.

PubMed

Lengerer, Birgit; Pjeta, Robert; Wunderer, Julia; Rodrigues, Marcelo; Arbore, Roberto; Schärer, Lukas; Berezikov, Eugene; Hess, Michael W; Pfaller, Kristian; Egger, Bernhard; Obwegeser, Sabrina; Salvenmoser, Willi; Ladurner, Peter

2014-02-12

Free-living flatworms, in both marine and freshwater environments, are able to adhere to and release from a substrate several times within a second. This reversible adhesion relies on adhesive organs comprised of three cell types: an adhesive gland cell, a releasing gland cell, and an anchor cell, which is a modified epidermal cell responsible for structural support. However, nothing is currently known about the molecules that are involved in this adhesion process. In this study we present the detailed morphology of the adhesive organs of the free-living marine flatworm Macrostomum lignano. About 130 adhesive organs are located in a horse-shoe-shaped arc along the ventral side of the tail plate. Each organ consists of exactly three cells, an adhesive gland cell, a releasing gland cell, and an anchor cell. The necks of the two gland cells penetrate the anchor cell through a common pore. Modified microvilli of the anchor cell form a collar surrounding the necks of the adhesive- and releasing glands, jointly forming the papilla, the outer visible part of the adhesive organs. Next, we identified an intermediate filament (IF) gene, macif1, which is expressed in the anchor cells. RNA interference mediated knock-down resulted in the first experimentally induced non-adhesion phenotype in any marine animal. Specifically, the absence of intermediate filaments in the anchor cells led to papillae with open tips, a reduction of the cytoskeleton network, a decline in hemidesmosomal connections, and to shortened microvilli containing less actin. Our findings reveal an elaborate biological adhesion system in a free-living flatworm, which permits impressively rapid temporary adhesion-release performance in the marine environment. We demonstrate that the structural integrity of the supportive cell, the anchor cell, is essential for this adhesion process: the knock-down of the anchor cell-specific intermediate filament gene resulted in the inability of the animals to adhere. The RNAi mediated changes of the anchor cell morphology are comparable to situations observed in human gut epithelia. Therefore, our current findings and future investigations using this powerful flatworm model system might contribute to a better understanding of the function of intermediate filaments and their associated human diseases.
Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase.

PubMed

Kil, In Sup; Huh, Tae Lin; Lee, Young Sup; Lee, You Mie; Park, Jeen-Woo

2006-01-01

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.
ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

PubMed Central

Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias

2016-01-01

The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265
The modeling of ethanol production by Kluyveromyces marxianus using whey as substrate in continuous A-Stat bioreactors.

PubMed

Gabardo, Sabrina; Pereira, Gabriela Feix; Rech, Rosane; Ayub, Marco Antônio Záchia

2015-09-01

We investigated the kinetics of whey bioconversion into ethanol by Kluyveromyces marxianus in continuous bioreactors using the "accelerostat technique" (A-stat). Cultivations using free and Ca-alginate immobilized cells were evaluated using two different acceleration rates (a). The kinetic profiles of these systems were modeled using four different unstructured models, differing in the expressions for the specific growth (μ) and substrate consumption rates (r s), taking into account substrate limitation and product inhibition. Experimental data showed that the dilution rate (D) directly affected cell physiology and metabolism. The specific growth rate followed the dilution rate (μ≈D) for the lowest acceleration rate (a = 0.0015 h(-2)), condition in which the highest ethanol yield (0.52 g g(-1)) was obtained. The highest acceleration rate (a = 0.00667 h(-2)) led to a lower ethanol yield (0.40 g g(-1)) in the system where free cells were used, whereas with immobilized cells ethanol yields increased by 23 % (0.49 g g(-1)). Among the evaluated models, Monod and Levenspiel combined with Ghose and Tyagi models were found to be more appropriate for describing the kinetics of whey bioconversion into ethanol. These results may be useful in scaling up the process for ethanol production from whey.
Deletion of RhoA in Progesterone Receptor-Expressing Cells Leads to Luteal Insufficiency and Infertility in Female Mice.

PubMed

El Zowalaty, Ahmed E; Li, Rong; Zheng, Yi; Lydon, John P; DeMayo, Francesco J; Ye, Xiaoqin

2017-07-01

Ras homolog gene family, member A (RhoA) is widely expressed throughout the female reproductive system. To assess its role in progesterone receptor-expressing cells, we generated RhoA conditional knockout mice RhoAd/d (RhoAf/f-Pgr-Cre+/-). RhoAd/d female mice had comparable mating activity, serum luteinizing hormone, prolactin, and estradiol levels and ovulation with control but were infertile with progesterone insufficiency, indicating impaired steroidogenesis in RhoAd/d corpus luteum (CL). RhoA was highly expressed in wild-type luteal cells and conditionally deleted in RhoAd/d CL. Gestation day 3.5 (D3.5) RhoAd/d ovaries had reduced numbers of CL, less defined corpus luteal cord formation, and disorganized CL collagen IV staining. RhoAd/d CL had lipid droplet and free cholesterol accumulation, indicating the availability of cholesterol for steroidogenesis, but disorganized β-actin and vimentin staining, indicating disrupted cytoskeleton integrity. Cytoskeleton is important for cytoplasmic cholesterol movement to mitochondria and for regulating mitochondria. Dramatically reduced expression of mitochondrial markers heat shock protein 60 (HSP60), voltage-dependent anion channel, and StAR was detected in RhoAd/d CL. StAR carries out the rate-limiting step of steroidogenesis. StAR messenger RNA expression was reduced in RU486-treated D3.5 wild-type CL and tended to be induced in progesterone-treated D3.5 RhoAd/d CL, with parallel changes of HSP60 expression. These data demonstrated the in vivo function of RhoA in CL luteal cell cytoskeleton integrity, cholesterol transport, StAR expression, and progesterone synthesis, and a positive feedback on StAR expression in CL by progesterone signaling. These findings provide insights into mechanisms of progesterone insufficiency.
Lysophosphatidic acid directly induces macrophage-derived foam cell formation by blocking the expression of SRBI.

PubMed

Chen, Linmu; Zhang, Jun; Deng, Xiao; Liu, Yan; Yang, Xi; Wu, Qiong; Yu, Chao

2017-09-23

The leading cause of morbidity and mortality is the result of cardiovascular disease, mainly atherosclerosis. The formation of macrophage foam cells by ingesting ox-LDL and focal retention in the subendothelial space are the hallmarks of the early atherosclerotic lesion. Lysophosphatidic acid (LPA), which is a low-molecular weight lysophospholipid enriched in oxidized LDL, exerts a range of effects on the cardiovascular system. Previous reports show that LPA increases the uptake of ox-LDL to promote the formation of foam cells. However, as the most active component of ox-LDL, there is no report showing whether LPA directly affects foam cell formation. The aim of this study was to investigate the effects of LPA on foam cell formation, as well as to elucidate the underlying mechanism. Oil red O staining and a Cholesterol/cholesteryl ester quantitation assay were used to evaluate foam cell formation in Raw264.7 macrophage cells. We utilized a Western blot and RT-PCR to investigate the relationship between LPA receptors and lipid transport related proteins. We found that LPA promoted foam cell formation, using 200 μM for 24 h. Meanwhile, the expression of the Scavenger receptor BI (SRBI), which promotes the efflux of free cholesterol, was decreased. Furthermore, the LPA 1/3 receptor antagonist Ki16425 significantly abolished the LPA effects, indicating that LPA 1/3 was involved in the foam cell formation and SRBI expression induced by LPA. Additionally, the LPA-induced foam cell formation was blocked with an AKT inhibitor. Our results suggest that LPA-enhanced foam cell formation is mediated by LPA 1/3 -AKT activation and subsequent SRBI expression. Copyright © 2017. Published by Elsevier Inc.
Hydrogen Exchange During Cell-Free Incorporation of Deuterated Amino Acids and an Approach to its Inhibition

PubMed Central

Tonelli, M.; Singarapu, K. K.; Markley, J. L.; Makino, S.; Sahu, S .C.; Matsubara, Y.; Endo, Y.; Kainosho, M.

2012-01-01

Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H2O, exchange reactions can lead to contamination of 2H sites by 1H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing 1H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM L-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-2H,15N]-chlorella ubiquitin without and with added inhibitors, and [U-15N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-13C,15N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at Cα sites with the exception of Gly. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Ala-Hβ, Asn-Hβ, Asp-Hβ, Gln-Hγ, Glu-Hγ, and Lys-Hε. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of interest in studies of large proteins, protein complexes, and membrane proteins. PMID:21984356
High Expression of KCa3.1 in Patients with Clear Cell Renal Carcinoma Predicts High Metastatic Risk and Poor Survival

PubMed Central

Rabjerg, Maj; Oliván-Viguera, Aida; Hansen, Lars Koch; Jensen, Line; Sevelsted-Møller, Linda; Walter, Steen; Jensen, Boye L.; Marcussen, Niels; Köhler, Ralf

2015-01-01

Background Ca2+-activated K+ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca2+-activated K+ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival. Methodology/Principal Findings We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro. Conclusions/Significance Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC. PMID:25848765
High expression of KCa3.1 in patients with clear cell renal carcinoma predicts high metastatic risk and poor survival.

PubMed

Rabjerg, Maj; Oliván-Viguera, Aida; Hansen, Lars Koch; Jensen, Line; Sevelsted-Møller, Linda; Walter, Steen; Jensen, Boye L; Marcussen, Niels; Köhler, Ralf

2015-01-01

Ca2+-activated K+ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca2+-activated K+ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival. We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro. Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC.
Refolding strategies from inclusion bodies in a structural genomics project.

PubMed

Trésaugues, Lionel; Collinet, Bruno; Minard, Philippe; Henckes, Gilles; Aufrère, Robert; Blondeau, Karine; Liger, Dominique; Zhou, Cong-Zhao; Janin, Joël; Van Tilbeurgh, Herman; Quevillon-Cheruel, Sophie

2004-01-01

The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.
Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum.

PubMed

Losurdo, Luca; Quintieri, Laura; Caputo, Leonardo; Gallerani, Raffaele; Mayo, Baltasar; De Leo, Francesca

2013-03-01

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 μg of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 μg of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Differential expression of α-L-arabinofuranosidases during maize (Zea mays L.) root elongation.

PubMed

Kozlova, Liudmila V; Gorshkov, Oleg V; Mokshina, Natalia E; Gorshkova, Tatyana A

2015-05-01

Specific α- l -arabinofuranosidases are involved in the realisation of elongation growth process in cells with type II cell walls. Elongation growth in a plant cell is largely based on modification of the cell wall. In type II cell walls, the Ara/Xyl ratio is known to decrease during elongation due to the partial removal of Ara residues from glucuronoarabinoxylan. We searched within the maize genome for the genes of all predicted α-L-arabinofuranosidases that may be responsible for such a process and related their expression to the activity of the enzyme and the amount of free arabinose measured in six zones of a growing maize root. Eight genes of the GH51 family (ZmaABFs) and one gene of the GH3 family (ZmaARA-I) were identified. The abundance of ZmaABF1 and 3-6 transcripts was highly correlated with the measured enzymatic activity and free arabinose content that significantly increased during elongation. The transcript abundances also coincided with the pattern of changes in the Ara/Xyl ratio of the xylanase-extractable glucuronoarabinoxylan described in previous studies. The expression of ZmaABF3, 5 and 6 was especially up-regulated during elongation although corresponding proteins are devoid of the catalytic glutamate at the proper position. ZmaABF2 transcripts were specifically enriched in the root cap and meristem. A single ZmaARA-I gene was not expressed as a whole gene but instead as splice variants that encode the C-terminal end of the protein. Changes in the ZmaARA-I transcript level were rather moderate and had no significant correlation with free arabinose content. Thus, elongation growth of cells with type II cell walls is accompanied by the up-regulation of specific and predicted α-L-arabinofuranosidase genes, and the corresponding activity is indeed pronounced and is important for the modification of glucuronoarabinoxylan, which plays a key role in the modification of the cell wall supramolecular organisation.
The 5'-untranslated region of p23 mRNA from the Ehrlich ascites tumor is involved in translation control of the growth related protein p23.

PubMed

Böhm, H; Gross, B; Gaestel, M; Bommer, U A; Ryffel, G; Bielka, H

1991-01-01

The growth-related protein p23 of the Ehrlich ascites tumor (EAT) is preferentially expressed in the exponentially growing tumor; its synthesis is translationally controlled. p23 mRNA is efficiently translated in the wheat germ cell-free lysate. In contrast, p23 mRNA present in poly(A)+RNA isolated from EAT is not translated in cell-free systems of EAT and reticulocytes. Moreover, translation of a p23 transcript is inhibited in the presence of total poly(A)+RNA. This inhibition is abolished by the removal of the 5'-UTR of the p23 transcript. Solution hybridization/RNase protection experiments point to the presence of a nucleotide sequence complementary to the 5'-UTR of p23 mRNA which might be involved in p23 mRNA inhibition.
Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

PubMed

Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

1978-09-01

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.
Which has more stem-cell characteristics: Müller cells or Müller cells derived from in vivo culture in neurospheres?

PubMed

Ji, Hong-Pei; Xiong, Yu; Zhang, En-Dong; Song, Wei-Tao; Gao, Zhao-Lin; Yao, Fei; Sun, Hong; Zhou, Rong-Rong; Xia, Xiao-Bo

2017-01-01

Müller cells can be acquired from in vitro culture or a neurosphere culture system. Both culture methods yield cells with progenitor-cell characteristics that can differentiate into mature nervous cells. We compared the progenitor-cell traits of Müller cells acquired from each method. Primary murine Müller cells were isolated in serum culture media and used to generate Müller cells derived from neurospheres in serum-free culture conditions. Gene expression of neural progenitor cell markers was examined by Q-PCR in the two groups. Expression of rhodopsin and the cone-rod homeobox protein CRX were assessed after induction with 1 μM all-trans retinoic acid (RA) for 7 days. After more than four passages, many cells were large, flattened, and difficult to passage. A spontaneously immortalized Müller cell line was not established. Three-passage neurospheres yielded few new spheres. Genes coding for Nestin, Sox2, Chx10, and Vimentin were downregulated in cells derived from neurospheres compared to the cells from standard culture, while Pax6 was upregulated. Müller cells from both culture methods were induced into rod photoreceptors, but expression of rhodopsin and CRX was greater in the Müller cells from the standard culture. Both culture methods yielded cells with stem-cell characteristics that can be induced into rod photoreceptor neurons by RA. Serum had no influence on the "stemness" of the cells. Cells from standard culture had greater "stemness" than cells derived from neurospheres. The standard Müller cells would seem to be the best choice for transplantation in cell replacement therapy for photoreceptor degeneration.
Role of NADPH oxidases in inducing a selective increase of oxidant stress and cyclin D1 and checkpoint 1 over-expression during progression to human gastric adenocarcinoma.

PubMed

Montalvo-Javé, Eduardo E; Olguín-Martínez, Marisela; Hernández-Espinosa, Diego R; Sánchez-Sevilla, Lourdes; Mendieta-Condado, Edgar; Contreras-Zentella, Martha L; Oñate-Ocaña, Luis F; Escalante-Tatersfield, Tomás; Echegaray-Donde, Agustín; Ruiz-Molina, Juan M; Herrera, Miguel F; Morán, Julio; Hernández-Muñoz, Rolando

2016-04-01

Gastric cancer is one of the main causes of global mortality. Here, reactive oxygen species (ROS) could largely contribute to gastric carcinogenesis. Hence, the present work was aimed to assess the role of ROS, oxidant status, NADPH oxidases (NOXs) expression, during human gastric adenocarcinoma. We obtained subcellular fraction from samples of gastric mucosa taken from control subjects (n = 20), and from 40 patients with gastric adenocarcinoma, as well as samples of distant areas (tumour-free gastric mucosa). Parameters indicative of lipid peroxidation and cell proliferation were selectively increased in both tumour-free and in cancerous gastric mucosa, despite of glutathione (GSH) content, glutathione reductase (GR) and superoxide dismutase (SOD) activities were increased in the adenocarcinoma. These high levels of antioxidant defences inversely correlated with down-regulated expression for NOX2 and 4; however, over-expression of NOX1 occurred with increased caspase-3 activity and overexpressed checkpoint 1 (MDC1) and cyclin D1 proteins. In the tumour-free mucosa an oxidant stress took place, without changing total GSH but with decreased activities for GR and mitochondrial SOD; moreover, over-expression of checkpoint 1 (MDC1) correlated with lower NOX2 and 4 expression in this mucosa. Chronically injured gastric mucosa increases lipoperoxidative events and cell proliferation. In the adenocarcinoma, cell proliferation was further enhanced, oxidant stress decreased which seemed to be linked to NOX1, MDC1 and cyclin D1 over-expression, but with a lower NOXs activity leading a 'low tone' of ROS formation. Therefore, our results could be useful for early detection and treatment of gastric adenocarcinoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemically defined serum-free conditions for cartilage regeneration from human embryonic stem cells.

PubMed

Yang, Dandan; Chen, Shubin; Gao, Changzhao; Liu, Xiaobo; Zhou, Yulai; Liu, Pengfei; Cai, Jinglei

2016-11-01

The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro, and test the effect of in vivo environments on the further maturation of hESCs derived cells. Embryoid bodies (EBs) formed from hESCs, with serum-free KSR-based medium and mesodermal specification related factors, CHIR, and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB, TGF β3, BMP4 in sequence for 24days. The morphology, FACS, histological staining as well as the expression of chondrogenic specific genes were detected in each stage, and further in vivo experiments, cell injections and tissue transplantations, further verified the formation of chondrocytes. We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule, their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes, and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease. Copyright © 2016 Elsevier Inc. All rights reserved.
Enhanced Expression of IL-18 and IL-18BP in Plasma of Patients with Eczema: Altered Expression of IL-18BP and IL-18 Receptor on Mast Cells.

PubMed

Hu, Yalin; Wang, Junling; Zhang, Huiyun; Xie, Hua; Song, Weiwei; Jiang, Qijun; Zhao, Nan; He, Shaoheng

2017-01-01

IL-18 has been found to be associated with eczema. However, little is known of the role of IL-18 binding protein (BP) and IL-18 receptor (R) in eczema. We therefore investigated the expression of IL-18, IL-18BP, and IL-18R on mast cells by using flow cytometry analysis and mouse eczema model. The results showed that plasma free IL-18 and free IL-18BP levels in eczema patients were higher than those in healthy controls. IL-18 provoked up to 3.1-fold increase in skin mast cells. IL-18 induced also an increase in IL-18BP+ mast cells, but a reduction of IL-18R+ mast cells in mouse eczema skin. It was found that house dust mite allergen Der p1 and egg allergen OVA induced upregulation of the expression of IL-18, IL-18BP, and IL-18R mRNAs in HMC-1 cells following 2 and 16 h incubation. In conclusion, correlation of IL-18 and IL-18BP in eczema plasma suggests an important balance between IL-18 and IL-18BP in eczema. The decrease in molar concentration ratio of plasma IL-18BP/IL-18 and allergen-induced upregulated expression of IL-18 and IL-18R in skin mast cells of the patients with eczema suggests that anti-IL-18 including IL-18BP therapy may be useful for the treatment of eczema.
Enhanced Expression of IL-18 and IL-18BP in Plasma of Patients with Eczema: Altered Expression of IL-18BP and IL-18 Receptor on Mast Cells

PubMed Central

2017-01-01

IL-18 has been found to be associated with eczema. However, little is known of the role of IL-18 binding protein (BP) and IL-18 receptor (R) in eczema. We therefore investigated the expression of IL-18, IL-18BP, and IL-18R on mast cells by using flow cytometry analysis and mouse eczema model. The results showed that plasma free IL-18 and free IL-18BP levels in eczema patients were higher than those in healthy controls. IL-18 provoked up to 3.1-fold increase in skin mast cells. IL-18 induced also an increase in IL-18BP+ mast cells, but a reduction of IL-18R+ mast cells in mouse eczema skin. It was found that house dust mite allergen Der p1 and egg allergen OVA induced upregulation of the expression of IL-18, IL-18BP, and IL-18R mRNAs in HMC-1 cells following 2 and 16 h incubation. In conclusion, correlation of IL-18 and IL-18BP in eczema plasma suggests an important balance between IL-18 and IL-18BP in eczema. The decrease in molar concentration ratio of plasma IL-18BP/IL-18 and allergen-induced upregulated expression of IL-18 and IL-18R in skin mast cells of the patients with eczema suggests that anti-IL-18 including IL-18BP therapy may be useful for the treatment of eczema. PMID:28839348
Inhibitory effect of emodin on fatty acid synthase, colon cancer proliferation and apoptosis.

PubMed

Lee, Kyung Ha; Lee, Myung Sun; Cha, Eun Young; Sul, Ji Young; Lee, Jin Sun; Kim, Jin Su; Park, Jun Beom; Kim, Ji Yeon

2017-04-01

Fatty acid synthase (FASN) is a key anabolic enzyme for de novo fatty acid synthesis, which is important in the development of colon carcinoma. The high expression of FASN is considered a promising molecular target for colon cancer therapy. Emodin, a naturally occurring anthraquinone, exhibits an anticancer effect in various types of human cancer, including colon cancer; however, the molecular mechanisms remain to be fully elucidated. Cell viability was evaluated using a Cell Counting Kit‑8 assay. The apoptosis rate of cells was quantified via flow cytometry following Annexin V/propidium iodide staining. FASN activity was measured by monitoring oxidation of nicotinamide adenine dinucleotide phosphate at a wavelength of 340 nm, and intracellular free fatty acid levels were detected using a Free Fatty Acid Quantification kit. Western blot analysis and reverse transcription‑polymerase chain reaction were used to detect target gene and protein expression. The present study was performed to investigate whether the gene expression of FASN and its enzymatic activity are regulated by emodin in a human colon cancer cell line. Emodin markedly inhibited the proliferation of HCT116 cells and a higher protein level of FASN was expressed, compared with that in SW480, SNU-C2A or SNU‑C5 cells. Emodin significantly downregulated the protein expression of FASN in HCT116 cells, which was caused by protein degradation due to elevated protein ubiquitination. Emodin also inhibited intracellular FASN enzymatic activity and reduced the levels of intracellular free fatty acids. Emodin enhanced antiproliferation and apoptosis in a dose‑ and time‑dependent manner. The combined treatment of emodin and cerulenin, a commercial FASN inhibitor, had an additive effect on these activities. Palmitate, the final product of the FASN reaction, rescued emodin‑induced viability and apoptosis. In addition, emodin altered FASN‑involved signaling pathways, including phosphatidylinositol 3-kinase/Akt and mitogen‑activated protein kinases/extracellular signal-regulated kinases 1/2. These results suggested that emodin-regulated cell growth and apoptosis were mediated by inhibiting FASN and provide a molecular basis for colon cancer therapy.
Inhibitory effect of emodin on fatty acid synthase, colon cancer proliferation and apoptosis

PubMed Central

Lee, Kyung Ha; Lee, Myung Sun; Cha, Eun Young; Sul, Ji Young; Lee, Jin Sun; Kim, Jin Su; Park, Jun Beom; Kim, Ji Yeon

2017-01-01

Fatty acid synthase (FASN) is a key anabolic enzyme for de novo fatty acid synthesis, which is important in the development of colon carcinoma. The high expression of FASN is considered a promising molecular target for colon cancer therapy. Emodin, a naturally occurring anthraquinone, exhibits an anticancer effect in various types of human cancer, including colon cancer; however, the molecular mechanisms remain to be fully elucidated. Cell viability was evaluated using a Cell Counting Kit-8 assay. The apoptosis rate of cells was quantified via flow cytometry following Annexin V/propidium iodide staining. FASN activity was measured by monitoring oxidation of nicotinamide adenine dinucleotide phosphate at a wavelength of 340 nm, and intracellular free fatty acid levels were detected using a Free Fatty Acid Quantification kit. Western blot analysis and reverse transcription-polymerase chain reaction were used to detect target gene and protein expression. The present study was performed to investigate whether the gene expression of FASN and its enzymatic activity are regulated by emodin in a human colon cancer cell line. Emodin markedly inhibited the proliferation of HCT116 cells and a higher protein level of FASN was expressed, compared with that in SW480, SNU-C2A or SNU-C5 cells. Emodin significantly downregulated the protein expression of FASN in HCT116 cells, which was caused by protein degradation due to elevated protein ubiquitination. Emodin also inhibited intracellular FASN enzymatic activity and reduced the levels of intracellular free fatty acids. Emodin enhanced antiproliferation and apoptosis in a dose- and time-dependent manner. The combined treatment of emodin and cerulenin, a commercial FASN inhibitor, had an additive effect on these activities. Palmitate, the final product of the FASN reaction, rescued emodin-induced viability and apoptosis. In addition, emodin altered FASN-involved signaling pathways, including phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinases/extracellular signal-regulated kinases 1/2. These results suggested that emodin-regulated cell growth and apoptosis were mediated by inhibiting FASN and provide a molecular basis for colon cancer therapy. PMID:28260110
The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells.

PubMed

Pastor, Marie; Johnen, Sandra; Harmening, Nina; Quiviger, Mickäel; Pailloux, Julie; Kropp, Martina; Walter, Peter; Ivics, Zoltán; Izsvák, Zsuzsanna; Thumann, Gabriele; Scherman, Daniel; Marie, Corinne

2018-06-01

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Dual Effects of Cell Free Supernatants from Lactobacillus acidophilus and Lactobacillus rhamnosus GG in Regulation of MMP-9 by Up-Regulating TIMP-1 and Down-Regulating CD147 in PMA- Differentiated THP-1 Cells

PubMed Central

Maghsood, Faezeh; Mirshafiey, Abbas; Farahani, Mohadese M.; Modarressi, Mohammad Hossein; Jafari, Parvaneh; Motevaseli, Elahe

2018-01-01

Objective Recent studies have reported dysregulated expression of matrix metalloproteinases (MMPs), especially MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, TIMP-2), and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in activated macrophages of patients with inflammatory diseases. Therefore, MMP-2, MMP-9, and their regulators may represent a new target for treatment of inflammatory diseases. Probiotics, which are comprised of lactic acid bacteria, have the potential to modulate inflammatory responses. In this experimental study, we investigated the anti-inflammatory effects of cell-free supernatants (CFS) from Lactobacillus acidophilus (L. acidophilus) and L. rhamnosus GG (LGG) in phorbol myristate acetate (PMA)-differentiated THP-1 cells. Materials and Methods In this experimental study, PMA-differentiated THP-1 cells were treated with CFS from L. acidophilus, LGG and uninoculated bacterial growth media (as a control). The expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were determined using real-time quantitative reverse transcription polymerase chain reaction (RT- PCR). The levels of cellular surface expression of CD147 were assessed by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography. Results Our results showed that CFS from both L. acidophilus and LGG significantly inhibited the gene expression of MMP-9 (P=0.0011 and P=0.0005, respectively), increased the expression of TIMP-1 (P<0.0001), decreased the cell surface expression of CD147 (P=0.0307 and P=0.0054, respectively), and inhibited the gelatinolytic activity of MMP-9 (P=0.0003 and P<0.0001, respectively) in PMA-differentiated THP-1 cells. Although, MMP-2 expression and activity and TIMP-2 expression remained unchanged. Conclusion Our results indicate that CFS from L. acidophilus and LGG possess anti-inflammatory properties and can modulate the inflammatory response. PMID:29105390
Molecular imaging of the biological effects of quercetin and quercetin-rich foods.

PubMed

Moskaug, Jan Øivind; Carlsen, Harald; Myhrstad, Mari; Blomhoff, Rune

2004-04-01

The human diet contains several thousands of organic plant molecules (i.e. phytochemicals), many of which have significant bioactivities. The specific physiological effects of these compounds are impossible to predict from in vitro studies using cell cultures and cell-free model systems. Nutrigenomics, which may be defined as the application of genomic tools to study the integrated effects of nutrients on gene regulation, however, holds great promise in increasing the understanding of how nutrients affect molecular events in an organism. Quercetin, a phytochemical belonging to the flavonoids, has antioxidant activities, inhibit protein kinases, inhibit DNA topoisomerases and regulate gene expression. The aim of the present review is to describe some of the many effects of quercetin, and how molecular imaging using transgenic reporter mice may serve as a tool to study the integrated influence of quercetin and other dietary phytochemicals on gene expression in vivo. We are using the bioluminescence emitted from firefly luciferase as the reporter since light originating from the inside of a cell or organism can be detected externally in an intact living organism. Molecular imaging using reporter models is therefore a unique technology to study the integrated effects of environmental insults and dietary substances on the influence of gene expression in disease development. We utilize these in vivo models to elucidate the role of various flavonoids, such as quercetin, for modulating gene expression related to oxidative stress and the antioxidant defence system.
Methamphetamine abuse affects gene expression in brain-derived microglia of SIV-infected macaques to enhance inflammation and promote virus targets.

PubMed

Najera, Julia A; Bustamante, Eduardo A; Bortell, Nikki; Morsey, Brenda; Fox, Howard S; Ravasi, Timothy; Marcondes, Maria Cecilia Garibaldi

2016-04-23

Methamphetamine (Meth) abuse is a major health problem linked to the aggravation of HIV- associated complications, especially within the Central Nervous System (CNS). Within the CNS, Meth has the ability to modify the activity/function of innate immune cells and increase brain viral loads. Here, we examined changes in the gene expression profile of neuron-free microglial cell preparations isolated from the brain of macaques infected with the Simian Immunodeficiency Virus (SIV), a model of neuroAIDS, and exposed to Meth. We aimed to identify molecular patterns triggered by Meth that could explain the detection of higher brain viral loads and the development of a pro-inflammatory CNS environment in the brain of infected drug abusers. We found that Meth alone has a strong effect on the transcription of genes associated with immune pathways, particularly inflammation and chemotaxis. Systems analysis led to a strong correlation between Meth exposure and enhancement of molecules associated with chemokines and chemokine receptors, especially CXCR4 and CCR5, which function as co-receptors for viral entry. The increase in CCR5 expression was confirmed in the brain in correlation with increased brain viral load. Meth enhances the availability of CCR5-expressing cells for SIV in the brain, in correlation with increased viral load. This suggests that Meth is an important factor in the susceptibility to the infection and to the aggravated CNS inflammatory pathology associated with SIV in macaques and HIV in humans.
GPR41/FFAR3 and GPR43/FFAR2 as cosensors for short-chain fatty acids in enteroendocrine cells vs FFAR3 in enteric neurons and FFAR2 in enteric leukocytes.

PubMed

Nøhr, Mark K; Pedersen, Maria H; Gille, Andreas; Egerod, Kristoffer L; Engelstoft, Maja S; Husted, Anna Sofie; Sichlau, Rasmus M; Grunddal, Kaare V; Poulsen, Steen Seier; Han, Sangdon; Jones, Robert M; Offermanns, Stefan; Schwartz, Thue W

2013-10-01

The expression of short-chain fatty acid receptors GPR41/FFAR3 and GPR43/ free fatty acid receptor 2 (FFAR2) was studied in the gastrointestinal tract of transgenic monomeric red fluorescent protein (mRFP) reporter mice. In the stomach free fatty acid receptor 3 (FFAR3)-mRFP was expressed in a subpopulation of ghrelin and gastrin cells. In contrast, strong expression of FFAR3-mRFP was observed in all cholecystokinin, glucose-dependent insulinotropic peptide (GIP), and secretin cells of the proximal small intestine and in all glucagon-like peptide-1 (GLP-1), peptide YY, and neurotensin cells of the distal small intestine. Throughout the colon and rectum, FFAR3-mRFP was strongly expressed in the large population of peptide YY and GLP-1 cells and in the neurotensin cells of the proximal colon. A gradient of expression of FFAR3-mRFP was observed in the somatostatin cells from less than 5% in the stomach to more than 95% in the rectum. Substance P-containing enterochromaffin cells displayed a similar gradient of FFAR3-mRFP expression throughout the small intestine. Surprisingly, FFAR3-mRFP was also expressed in the neuronal cells of the submucosal and myenteric ganglia. Quantitative PCR analysis of fluorescence-activated cell sorting (FACS) purified FFAR3-mRFP positive cells confirmed the coexpression with the various peptide hormones as well as key neuronal marker proteins. The FFAR2-mRFP reporter was strongly expressed in a large population of leukocytes in the lamina propria of in particular the small intestine but surprisingly only weakly in a subpopulation of enteroendocrine cells. Nevertheless, synthetic ligands specific for either FFAR3 or FFAR2 each released GLP-1 from colonic crypt cultures and the FFAR2 agonist mobilized intracellular Ca²⁺ in FFAR2 positive enteroendocrine cells. It is concluded that FFAR3-mRFP serves as a useful marker for the majority of enteroendocrine cells of the small and large intestine and that FFAR3 and FFAR2 both act as sensors for short-chain fatty acids in enteroendocrine cells, whereas FFAR3 apparently has this role alone in enteric neurons and FFAR2 in enteric leukocytes.
The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease

PubMed Central

Sameni, Sara; Syed, Adeela; Marsh, J. Lawrence; Digman, Michelle A.

2016-01-01

Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening. PMID:27713486
The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease

NASA Astrophysics Data System (ADS)

Sameni, Sara; Syed, Adeela; Marsh, J. Lawrence; Digman, Michelle A.

2016-10-01

Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening.
Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity.

PubMed

Tauber, Svantje; Lauber, Beatrice A; Paulsen, Katrin; Layer, Liliana E; Lehmann, Martin; Hauschild, Swantje; Shepherd, Naomi R; Polzer, Jennifer; Segerer, Jürgen; Thiel, Cora S; Ullrich, Oliver

2017-01-01

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable "steady state" after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions.
Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity

PubMed Central

Tauber, Svantje; Lauber, Beatrice A.; Paulsen, Katrin; Layer, Liliana E.; Lehmann, Martin; Hauschild, Swantje; Shepherd, Naomi R.; Polzer, Jennifer; Segerer, Jürgen; Thiel, Cora S.

2017-01-01

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC–TOF–MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface–bound fucose. The reduced ICAM-1 expression and the loss of cell surface–bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable “steady state” after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions. PMID:28419128
Low oxygen tension enhances endothelial fate of human pluripotent stem cells.

PubMed

Kusuma, Sravanti; Peijnenburg, Elizabeth; Patel, Parth; Gerecht, Sharon

2014-04-01

A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor β+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.
An integrated systems genetics screen reveals the transcriptional structure of inherited predisposition to metastatic disease

PubMed Central

Faraji, Farhoud; Hu, Ying; Wu, Gang; Goldberger, Natalie E.; Walker, Renard C.; Zhang, Jinghui; Hunter, Kent W.

2014-01-01

Metastasis is the result of stochastic genomic and epigenetic events leading to gene expression profiles that drive tumor dissemination. Here we exploit the principle that metastatic propensity is modified by the genetic background to generate prognostic gene expression signatures that illuminate regulators of metastasis. We also identify multiple microRNAs whose germline variation is causally linked to tumor progression and metastasis. We employ network analysis of global gene expression profiles in tumors derived from a panel of recombinant inbred mice to identify a network of co-expressed genes centered on Cnot2 that predicts metastasis-free survival. Modulating Cnot2 expression changes tumor cell metastatic potential in vivo, supporting a functional role for Cnot2 in metastasis. Small RNA sequencing of the same tumor set revealed a negative correlation between expression of the Mir216/217 cluster and tumor progression. Expression quantitative trait locus analysis (eQTL) identified cis-eQTLs at the Mir216/217 locus, indicating that differences in expression may be inherited. Ectopic expression of Mir216/217 in tumor cells suppressed metastasis in vivo. Finally, small RNA sequencing and mRNA expression profiling data were integrated to reveal that miR-3470a/b target a high proportion of network transcripts. In vivo analysis of Mir3470a/b demonstrated that both promote metastasis. Moreover, Mir3470b is a likely regulator of the Cnot2 network as its overexpression down-regulated expression of network hub genes and enhanced metastasis in vivo, phenocopying Cnot2 knockdown. The resulting data from this strategy identify Cnot2 as a novel regulator of metastasis and demonstrate the power of our systems-level approach in identifying modifiers of metastasis. PMID:24322557
Stimulatory effect of insulin on 5alpha-reductase type 1 (SRD5A1) expression through an Akt-dependent pathway in ovarian granulosa cells.

PubMed

Kayampilly, Pradeep P; Wanamaker, Brett L; Stewart, James A; Wagner, Carrie L; Menon, K M J

2010-10-01

Elevated levels of 5α-reduced androgens have been shown to be associated with hyperandrogenism and hyperinsulinemia, the leading causes of ovulatory dysfunction in women. 5α-Dihydrotestosterone reduces ovarian granulosa cell proliferation by inhibiting FSH-mediated mitogenic signaling pathways. The present study examined the effect of insulin on 5α-reductase, the enzyme that catalyses the conversion of androgens to their 5α-derivatives. Granulosa cells isolated from immature rat ovaries were cultured in serum-free, phenol red-free DMEM-F12 media and treated with different doses of insulin (0, 0.1, 1.0, and 10.0 μg/ml) for different time intervals up to 12 h. The expression of 5α-reductase type 1 mRNA, the predominant isoform found in granulosa cells, showed a significant (P<0.05) increase in response to the insulin treatment up to 12 h compared with control. The catalytic activity of 5α-reductase enzyme was also stimulated in a dose-depended manner (P<0.05). Inhibiting the Akt-dependent signaling pathway abolished the insulin-mediated increase in 5α-reductase mRNA expression, whereas inhibition of the ERK-dependent pathway had no effect. The dose-dependent increase in 5α-reductase mRNA expression as well as catalytic activity seen in response to insulin treatment was also demonstrated in the human granulosa cell line (KGN). In addition to increased mRNA expression, a dose-dependent increase in 5α-reductase protein expression in response to insulin was also seen in KGN cells, which corroborated well with that of mRNA expression. These results suggest that elevated levels of 5α-reduced androgens seen in hyperinsulinemic conditions might be explained on the basis of a stimulatory effect of insulin on 5α-reductase in granulosa cells. The elevated levels of these metabolites, in turn, might adversely affect growth and proliferation of granulosa cells, thereby impairing follicle growth and ovulation.
Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

PubMed

No, Jin-Gu; Choi, Mi-Kyung; Kwon, Dae-Jin; Yoo, Jae Gyu; Yang, Byoung-Chul; Park, Jin-Ki; Kim, Dong-Hoon

2015-01-01

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot(TM) reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.
Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

NASA Technical Reports Server (NTRS)

Parks, Kelsey

2009-01-01

Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.
Hepcidin as a possible marker in determination of malignancy degree and sensitivity of breast cancer cells to cytostatic drugs.

PubMed

Yalovenko, T M; Todor, I M; Lukianova, N Y; Chekhun, V F

2016-06-01

To investigate the role of hepcidin (Hepc) in the formation of cells malignant phenotype in vitro and its expression in the dyna-mics of growth of Walker-256 carcinosarcoma with different sensitivity to doxorubicin (Dox). The cell lines used in the analysis included T47D, MCF-7, MDA-MB-231, MDA-MB-468, MCF/CP, and MCF/Dox. Hepc expression was studied by immunocytochemical method. "Free" iron content was determined by EPR spectroscopy. Determination of Hepc expression in homogenates of tumor tissue and in blood serum of rats with Dox-sensitive and -resistant Walker-256 carcinosarcoma was performed. It was found that Hepc levels in breast cancer (BC) cells with high degree of malignancy (MDA-MB-231, MDA-MB-468) and drug-resistant phenotype (MCF/CP, MCF/Dox) were by 1.5-2 times higher (p < 0.05) in comparison with sensitive and less malignant BC cells. The development of drug-resistant phenotype in Walker-256 carcinosarcoma cells was accompanied by increasing of Hepc and "free" iron content (by 2.4 and 1.2 times, respectively). The data of in vitro and in vivo research evidenced on involvement of Hepc in formation of BC cells malignant phenotype and their resistance to Dox.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

PubMed

Meraviglia, Viviana; Zanon, Alessandra; Lavdas, Alexandros A; Schwienbacher, Christine; Silipigni, Rosamaria; Di Segni, Marina; Chen, Huei-Sheng Vincent; Pramstaller, Peter P; Hicks, Andrew A; Rossini, Alessandra

2015-06-05

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.
Overexpression of stathmin1 in the diffuse type of gastric cancer and its roles in proliferation and migration of gastric cancer cells.

PubMed

Jeon, T-Y; Han, M-E; Lee, Y-W; Lee, Y-S; Kim, G-H; Song, G-A; Hur, G-Y; Kim, J-Y; Kim, H-J; Yoon, S; Baek, S-Y; Kim, B-S; Kim, J-B; Oh, S-O

2010-02-16

Stathmin1 is a microtubule-regulating protein that has an important role in the assembly and disassembly of the mitotic spindle. The roles of stathmin1 in carcinogenesis of various cancers, including prostate and breast cancer, have been explored. However, its expression and roles in gastric cancer have not yet been described. Stathmin1 expression in paraffin-embedded tissue sections from 226 patients was analysed by immunohistochemistry. Roles of stathmin1 were studied using a specific small interfering RNA (siRNA). The expression of stathmin1 was positively correlated with lymph node metastasis, TNM stages and vascular invasion, and negatively with recurrence-free survival, in the diffuse type of gastric cancer. The median recurrence-free survival in patients with a negative and positive expression of stathmin1 was 17.0 and 7.0 months, respectively (P=0.009). When the expression of stathmin1 was knocked down using siRNA, the proliferation, migration and invasion of poorly differentiated gastric cancer cells in vitro were significantly inhibited. Moreover, stathmin1 siRNA transfection significantly slowed the growth of xenografts in nude mice. These results suggest that stathmin1 can be a good prognostic factor for recurrence-free survival rate and is a therapeutic target in diffuse-type gastric cancer.
PAI-1 expression and its regulation by promoter 4G/5G polymorphism in clear cell renal cell carcinoma.

PubMed

Choi, Jung-Woo; Lee, Ju-Han; Park, Hong Seok; Kim, Young-Sik

2011-10-01

To characterise patients with high plasminogen activator inhibitor-1 (PAI-1) expression as oral PAI-1 antagonists are currently in preclinical trials, and to determine whether the PAI-1 promoter 4G/5G polymorphism regulates PAI-1 expression in clear cell renal cell carcinoma (CCRCC). PAI-1 expression was examined by immunohistochemistry in 69 CCRCC specimens. In addition, the promoter 4G/5G polymorphism was investigated by both allele-specific PCR and direct DNA sequencing. PAI-1 was overexpressed in 25/69 (36.2%) patients with CCRCC. PAI-1 staining was intense in tumour cells with a high Fuhrman nuclear grade and in spindle-shaped tumour cells. PAI-1 expression was significantly associated with older age at diagnosis (p=0.027), high nuclear grade (p<0.001), advanced clinical stage (p=0.030) and distant metastasis (p=0.009). In survival analyses, PAI-1 expression was correlated with disease-free survival in Kaplan-Meier curves (p=0.015) but was not significant in the Cox hazards model (p=0.527). The frequencies of the promoter polymorphism were 24.6% (17/69) 4G/4G, 43.5% (30/69) 4G/5G and 31.9% (22/69) 5G/5G. The homozygous 4G/4G or 5G/5G group showed a tendency for a high nuclear grade (p=0.05) but the 4G/5G polymorphism was not related to other prognostic parameters. PAI-1 expression was poorly correlated with its promoter 4G/5G polymorphism (Spearman ρ=0.088). CCRCC with high PAI-1 expression is characterised by older age, high nuclear grade, advanced stage, distant metastasis and/or shortened disease-free survival. PAI-1 expression is not affected by the promoter 4G/5G polymorphism.
Expression of Lectins in Heterologous Systems

PubMed Central

Martínez-Alarcón, Dania; Blanco-Labra, Alejandro

2018-01-01

Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, there is great interest in their production on a large scale. Unfortunately, conventional techniques do not provide the appropriate platform for this purpose and therefore, the heterologous production of lectins in different organisms has become the preferred method in many cases. Such systems have the advantage of providing better yields as well as more homogeneous and better-defined properties for the resultant products. However, an inappropriate choice of the expression system can cause important structural alterations that have repercussions on their biological activity since the specificity may lay in their post-translational processing, which depends largely on the producing organism. The present review aims to examine the most representative studies in the area, exposing the four most frequently used systems (bacteria, yeasts, plants and animal cells), with the intention of providing the necessary information to determine the strategy to follow in each case as well as their respective advantages and disadvantages. PMID:29466298
Head and neck cancer relapse after chemoradiotherapy correlates with CD163+ macrophages in primary tumour and CD11b+ myeloid cells in recurrences.

PubMed

Balermpas, P; Rödel, F; Liberz, R; Oppermann, J; Wagenblast, J; Ghanaati, S; Harter, P N; Mittelbronn, M; Weiss, C; Rödel, C; Fokas, E

2014-10-14

We investigated the prognostic role of tumour-associated macrophages (TAMs) in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT). The expression of CD68+, CD163+ and CD11b+ cells was assessed using immunohistochemistry in n=106 pre-treatment tumour biopsy samples and was correlated with clinicopathological characteristics, including T-stage, N-stage, grading, tumour localisation, age and sex as well as local failure-free survival (LFFS), distant metastases-free survival (DMFS), progression-free (PFS), and overall survival (OS). Finally, TAMs expression and vessel density (CD31) were examined in n=12 available early local recurrence samples and compared with their matched primary tumours . The diagnostic images and radiotherapy plans of these 12 patients were also analysed. All local recurrences occurred in the high radiation dose region (⩾70 Gy). With a median follow-up of 40 months, OS at 2 years was 60.5%. High CD163 expression in primary tumours was associated with decreased OS (P=0.010), PFS (P=0.033), LFFS (P=0.036) and DMFS (P=0.038) in multivariate analysis. CD163 demonstrated a strong prognostic value only in human papillomavirus (p16(INK4))-negative patients. Early local recurrence specimens demonstrated a significantly increased infiltration of CD11b+ myeloid cells (P=0.0097) but decreased CD31-positive vessel density (P=0.0004) compared with their matched primary samples. Altogether, baseline CD163 expression predicts for an unfavourable clinical outcome in HNSCC after definitive CRT. Early local recurrences showed increased infiltration by CD11b+ cells. These data provide important insight on the role of TAMs in mediating response to CRT in patients with HNSCC.
Coupling of Peripheral Tolerance to Endogenous Interleukin 10 Promotes Effective Modulation of Myelin-Activated T Cells and Ameliorates Experimental Allergic Encephalomyelitis

PubMed Central

Legge, Kevin L.; Min, Booki; Bell, J. Jeremiah; Caprio, Jacque C.; Li, Lequn; Gregg, Randal K.; Zaghouani, Habib

2000-01-01

Several immune-based approaches are being considered for modulation of inflammatory T cells and amelioration of autoimmune diseases. The most recent strategies include simulation of peripheral self-tolerance by injection of adjuvant free antigen, local delivery of cytokines by genetically altered T cells, and interference with the function of costimulatory molecules. Although promising results have been obtained from these studies that define mechanisms of T cell modulation, efficacy, practicality, and toxicity, concerns remain unsolved, thereby justifying further investigations to define alternatives for effective downregulation of aggressive T cells. In prior studies, we demonstrated that an immunoglobulin (Ig) chimera carrying the encephalitogenic proteolipid protein (PLP)1 peptide corresponding to amino acid sequence 139–151 of PLP, Ig-PLP1, is presented to T cells ∼100-fold better than free PLP1. Here, we demonstrate that aggregation endows Ig-PLP1 with an additional feature, namely, induction of interleukin (IL)-10 production by macrophages and dendritic cells, both of which are antigen-presenting cells (APCs). These functions synergize in vivo and drive effective modulation of autoimmunity. Indeed, it is shown that animals with ongoing active experimental allergic encephalomyelitis dramatically reduce the severity of their paralysis when treated with adjuvant free aggregated Ig-PLP1. Moreover, IL-10 displays bystander antagonism on unrelated autoreactive T cells, allowing for reversal of disease involving multiple epitopes. Therefore, aggregated Ig-PLP1 likely brings together a peripheral T cell tolerance mechanism emanating from peptide presentation by APCs expressing suboptimal costimulatory molecules and IL-10 bystander suppression to drive a dual-modal T cell modulation system effective for reversal of autoimmunity involving several epitopes and diverse T cell specificities. PMID:10859329
[The cultivation and identification of lacrimal gland adenoid cystic cancer stem cells].

PubMed

Lyu, Jianmei; He, Yanjin; Xie, Lianfeng; Liu, Xun; Zhu, Limin

2015-10-01

To isolate and cultivate the Lacrimal gland Adenoid Cystic Carcinoma cells line, study Cancer Stem Cells properties. Experimental study. Lacrimal gland adenoid cystic carcinoma cancer stem cells were cultivated in serum-free suspension culture and the morphological changes were observed. Cells were divided into two groups, the LACC-CSC experimental group and the LACC control group. The flow cytometry instrument was used to detect the expression of classical stem cell markers CD133 and ABCG2. Transwell chamber was used to detect the cancer stem cell aggressivity and differentiated into the vascular endothelial cells. The tumorigenic force in vitro xenotransplantation were applied. LACC cells can grow suspensively after vaccinated in serum free medium and form tumor microspheres after 10-12 days. Flow cytometry experiments showed that the expression ratio of stem cell markers CD133 in LACC-CSC was (35.67 ± 6.86)%, significantly different to LACC with (0.46 ± 0.48)%, (t = 8.867, P < 0.05). Similarly, the expression ratio of stem cell marker ABCG2 in LACC-CSC was (39.99 ± 4.54)%, significantly different to LACC with (6.75 ± 1.34)%, (t = -9.932, P < 0.05). In vitro experiment of Matrigel invasion, LACC-CSC went through the matrigel basement membrane averagely (32.60 ± 8.79)/HP contrary to LACC with average (10.20 ± 2.77)/HP after 24 hours, showing statistically significance (t = 5.433, P < 0.05) between the two groups. After training for 48 hours, the difference between two groups was still obvious (t = 5.779, P < 0.05) with LACC-CSC average (62.60 ± 4.83)/HP to LACC (44.00 ± 5.34)/HP. When induced by serum medium containing VEGF and bFGF, LACC-CSC grew adherent gradually and cell morphological changes occurred after continuous induction to long spindle cells. When cultured into three-dimensional matrix structure they formed vessel samples and expressed vascular endothelial marker CD31 and CD34. Transplanted tumor in vitro experiment, mice of LACC-CSC group grew tumors in 9 days with 100% tumorigenic rate, whereas LACC group 12 days with 100% tumorigenic rate. LACC-CSC can be obtained through serum-free culture method. LACC-CSC grew suspensively and expressed classical stem cell markers. LACC-CSC were identified as cancer stem cells with stronger migration and invasion. LACC-CSC have tumorigenic force and multi-directional differentiation potential with general characteristics of the stem cell.
Heme-mediated cell activation: the inflammatory puzzle of sickle cell anemia.

PubMed

Guarda, Caroline Conceição da; Santiago, Rayra Pereira; Fiuza, Luciana Magalhães; Aleluia, Milena Magalhães; Ferreira, Júnia Raquel Dutra; Figueiredo, Camylla Vilas Boas; Yahouedehou, Setondji Cocou Modeste Alexandre; Oliveira, Rodrigo Mota de; Lyra, Isa Menezes; Gonçalves, Marilda de Souza

2017-06-01

Hemolysis triggers the onset of several clinical manifestations of sickle cell anemia (SCA). During hemolysis, heme, which is derived from hemoglobin (Hb), accumulates due to the inability of detoxification systems to scavenge sufficiently. Heme exerts multiple harmful effects, including leukocyte activation and migration, enhanced adhesion molecule expression by endothelial cells and the production of pro-oxidant molecules. Area covered: In this review, we describe the effects of heme on leukocytes and endothelial cells, as well as the features of vascular endothelial cells related to vaso-occlusion in SCA. Expert commentary: Free Hb, heme and iron, potent cytotoxic intravascular molecules released during hemolysis, can exacerbate, modulate and maintain the inflammatory response, a main feature of SCA. Endothelial cells in the vascular environment, as well as leukocytes, can become activated via the molecular signaling effects of heme. Due to the hemolytic nature of SCA, hemolysis represents an interesting therapeutic target for heme-scavenging purposes.
Betanodavirus Induces Oxidative Stress-Mediated Cell Death That Prevented by Anti-Oxidants and Zfcatalase in Fish Cells

PubMed Central

Chang, Chih-Wei; Su, Yu-Chin; Her, Guor-Mour; Ken, Chuian-Fu; Hong, Jiann-Ruey

2011-01-01

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12–24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24–48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection. Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis. PMID:21991373
Mesoporous silica nanoparticles loading doxorubicin reverse multidrug resistance: performance and mechanism

NASA Astrophysics Data System (ADS)

Shen, Jianan; He, Qianjun; Gao, Yu; Shi, Jianlin; Li, Yaping

2011-10-01

Multidrug resistance (MDR) is one of the major obstacles for successful chemotherapy in cancer. One of the effective approaches to overcome MDR is to use nanoparticle-mediated drug delivery to increase drug accumulation in drug resistant cancer cells. In this work, we first report that the performance and mechanism of an inorganic engineered delivery system based on mesoporous silica nanoparticles (MSNs) loading doxorubicin (DMNs) to overcome the MDR of MCF-7/ADR (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The experimental results showed that DMNs could enhance the cellular uptake of doxorubicin (DOX) and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells. The IC50 of DMNs against MCF-7/ADR cells was 8-fold lower than that of free DOX. However, an improved effect of DOX in DMNs against MCF-7 cells (a DOX-sensitive cancer cell line) was not found. The increased cellular uptake and nuclear accumulation of DOX delivered by DMNs in MCF-7/ADR cells was confirmed by confocal laser scanning microscopy, and could result from the down-regulation of P-gp and bypassing the efflux action by MSNs themselves. The cellular uptake mechanism of DMNs indicated that the macropinocytosis was one of the pathways for the uptake of DMNs by MCF-7/ADR cells. The in vivo biodistribution showed that DMNs induced a higher accumulation of DOX in drug resistant tumors than free DOX. These results suggested that MSNs could be an effective delivery system to overcome multidrug resistance.
Lymphocyte-conditioned medium in combination with interleukin-2 effectively induces antitumour autoimmunity by adoptive transfer of short activated killer (SHAK) cells.

PubMed

Buer, J; Hilse, R; Dallmann, I; Grosse, J; Kirchner, H; Zorn, U; Hänninen, E L; Franzke, A; Duensing, S; Poliwoda, H

1995-03-01

In this study, effective antitumour immunity was transferred by autologous short activated killer (SHAK) cells induced over four hours with lymphocyte conditioned medium (LCM) and recombinant interleukin-2 (rIL-2). Among eight patients with progressive metastatic renal cell carcinoma refractory to standard therapy, there were six objective tumour responses to SHAKs. Progression-free survival ranged from 0 to 8+ months, and overall survival ranged from 2 to 14+ months, with a median of 9+ months. Systemic toxicity of SHAKs was limited to flulike symptoms. Patient SHAKs provided a tumour-specific immunity, both cellular and humoral (expression and secretion of secondary cytokines, including IL-2, GM-CSF, INF-gamma and TNF-alpha), far superior to rIL-2 activated killer cells.
Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system.

PubMed

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

2012-09-01

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

PubMed

Yakimovich, Artur; Gumpert, Heidi; Burckhardt, Christoph J; Lütschg, Verena A; Jurgeit, Andreas; Sbalzarini, Ivo F; Greber, Urs F

2012-09-01

Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling.
Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation1[OPEN

PubMed Central

Yu, Peng; Eggert, Kai; von Wirén, Nicolaus; Li, Chunjian; Hochholdinger, Frank

2015-01-01

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box proteinS-Phase Kinase-Associated Protein 2B-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses. PMID:26198256
Adenosine decreases oxidative stress and protects H2O2-treated neural stem cells against apoptosis through decreasing Mst1 expression.

PubMed

Gholinejad, Masoumeh; Jafari Anarkooli, Iraj; Taromchi, Amirhossein; Abdanipour, Alireza

2018-05-01

Overproduction of free radicals during oxidative stress induces damage to key biomolecules and activates programed cell death pathways. Neuronal cell death in the nervous system leads to a number of neurodegenerative diseases. The aim of the present study was to evaluate the neuroprotective effect of adenosine on inhibition of apoptosis induced by hydrogen peroxide (H 2 O 2 ) in bone marrow-derived neural stem cells (B-dNSCs), with focus on its regulatory effect on the expression of mammalian sterile 20-like kinase 1 ( Mst1 ), as a novel proapoptotic kinase. B-dNSCs were exposed to adenosine at different doses (2, 4, 6, 8 and 10 µM) for 48 h followed by 125 µM H 2 O 2 for 30 min. Using MTT, terminal deoxynucleotidyl transferase dUTP nick-end labeling and real-time reverse transcription polymerase chain reaction assays, the effects of adenosine on cell survival, apoptosis and Mst1 , nuclear factor (erythroid-derived 2)-like 2 and B-cell lymphoma 2 and adenosine A1 receptor expression were evaluated in pretreated B-dNSCs compared with controls (cells treated with H 2 O 2 only). Firstly, results of the MTT assay indicated 6 µM adenosine to be the most protective dose in terms of promotion of cell viability. Subsequent assays using this dosage indicated that apoptosis rate and Mst1 expression in B-dNSCs pretreated with 6 µM adenosine were significantly decreased compared with the control group. These findings suggest that adenosine protects B-dNSCs against oxidative stress-induced cell death, and therefore, that it may be used to promote the survival rate of B-dNSCs and as a candidate for the treatment of oxidative stress-mediated neurological diseases.
The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis

PubMed Central

2014-01-01

Background It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. Results We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. Conclusions In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae. PMID:24602211
The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis.

PubMed

Koziol, Uriel; Rauschendorfer, Theresa; Zanon Rodríguez, Luis; Krohne, Georg; Brehm, Klaus

2014-03-06

It is believed that in tapeworms a separate population of undifferentiated cells, the germinative cells, is the only source of cell proliferation throughout the life cycle (similar to the neoblasts of free living flatworms). In Echinococcus multilocularis, the metacestode larval stage has a unique development, growing continuously like a mass of vesicles that infiltrate the tissues of the intermediate host, generating multiple protoscoleces by asexual budding. This unique proliferation potential indicates the existence of stem cells that are totipotent and have the ability for extensive self-renewal. We show that only the germinative cells proliferate in the larval vesicles and in primary cell cultures that undergo complete vesicle regeneration, by using a combination of morphological criteria and by developing molecular markers of differentiated cell types. The germinative cells are homogeneous in morphology but heterogeneous at the molecular level, since only sub-populations express homologs of the post-transcriptional regulators nanos and argonaute. Important differences are observed between the expression patterns of selected neoblast marker genes of other flatworms and the E. multilocularis germinative cells, including widespread expression in E. multilocularis of some genes that are neoblast-specific in planarians. Hydroxyurea treatment results in the depletion of germinative cells in larval vesicles, and after recovery following hydroxyurea treatment, surviving proliferating cells grow as patches that suggest extensive self-renewal potential for individual germinative cells. In E. multilocularis metacestodes, the germinative cells are the only proliferating cells, presumably driving the continuous growth of the larval vesicles. However, the existence of sub-populations of the germinative cells is strongly supported by our data. Although the germinative cells are very similar to the neoblasts of other flatworms in function and in undifferentiated morphology, their unique gene expression pattern and the evolutionary loss of conserved stem cells regulators suggest that important differences in their physiology exist, which could be related to the unique biology of E. multilocularis larvae.
Dental Pulp Stem Cell-Derived, Scaffold-Free Constructs for Bone Regeneration.

PubMed

Tatsuhiro, Fukushima; Seiko, Tatehara; Yusuke, Takebe; Reiko, Tokuyama-Toda; Kazuhito, Satomura

2018-06-22

In the present study, a scaffold-free tissue construct was developed as an approach for the regeneration of tissue defects, which produced good outcomes. We fabricated a scaffold-free tissue construct from human dental pulp stem cells (hDPSCs construct), and examined the characteristics of the construct. For its fabrication, basal sheets prepared by 4-week hDPSCs culturing were subjected to 1-week three-dimensional culture, with or without osteogenic induction, whereas hDPSC sheets (control) were fabricated by 1-week culturing of basal sheets on monolayer culture. The hDPSC constructs formed a spherical structure and calcified matrix that are absent in the control. The expression levels for bone-related genes in the hDPSC constructs were significantly upregulated compared with those in the control. Moreover, the hDPSC constructs with osteogenic induction had a higher degree of calcified matrix formation, and higher expression levels for bone-related genes, than those for the hDPSC constructs without osteogenic induction. These results suggest that the hDPSC constructs with osteogenic induction are composed of cells and extracellular and calcified matrices, and that they can be a possible scaffold-free material for bone regeneration.
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.
Effects of guanosine tetraphosphate on cell-free synthesis of Escherichia coli ribosomal RNA and other gene products.

PubMed Central

Reiness, G; Yang, H L; Zubay, G; Cashel, M

1975-01-01

A cell-free system derived from E. coli is described in which mature-sized 16S and 23S ribosomal RNAs (rRNA) are synthesized at a high relative rate, comprising 17-25% of the total transcription. The addition of guanosine tetraphosphate (ppGpp) to this system results in up to a 5-fold selective inhibition of rRNA accumulation. This effect is exerted at the level of synthesis rather than degradation. It is concluded that ppGpp, which is produced in large amounts by E. coli during amino-acid deprivation, could mediate the decrease in rRNA synthesis that accompanies such deprivation. The expression of other genes has also been investigated. No selective reduction of transfer RNA synthesis by ppGpp is observed. The trp and lac operons are found to be stimulated at the transcriptional level by the presence of this nucleotide. It is hypothesized that ppGpp interacts with the RNA polymerase in such a manner as to alter the affinity of the enzyme for promoters in an operon-specific fashion. PMID:1103124

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