National Center for Biotechnology

Madrid, Spain

National Center for Biotechnology

Madrid, Spain

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Tuleuova N.,University of California at Davis | Tuleuova N.,National Center for Biotechnology | Revzin A.,University of California at Davis
Cellular and Molecular Bioengineering | Year: 2010

Aptamer beacons are DNA or RNA probes that bind proteins or small molecules of interest and emit signal directly upon interaction with the target analyte. This paper describes micropatterning of aptamer beacons for detection of IFN-γ-an important inflammatory cytokine. The beacon consisted of a fluorophore-labeled aptamer strand hybridized with a shorter, quencher-carrying complementary strand. Cytokine molecules were expected to displace quenching strands of the beacon, disrupting FRET effect and resulting in fluorescence signal. The glass substrate was first micropatterned with poly(ethylene glycol) (PEG) hydrogel microwells (35 μm diameter individual wells) so as to define sites for attachment of beacon molecules. PEG microwell arrays were then incubated with avidin followed by biotin-aptamerfluorophore constructs. Subsequent incubation with quencher-carrying complementary strands resulted in formation of DNA duplex and caused quenching of fluorescence due to FRET effect. When exposed to IFN-γ, microwells changed fluorescence from low (quencher hybridized with fluorophore-carrying strand) to high (quenching strand displaced by cytokine molecules). The fluorescence signal was confined to microwells, was changing in real-time and was dependent on the concentration of IFN-γ. In the future, we plan to co-localize aptamer beacons and cells on micropatterned surfaces in order to monitor in real-time cytokine secretion from immune cells in microwells. © 2010 The Author(s).


Liu Y.,University of California at Davis | Tuleouva N.,University of California at Davis | Tuleouva N.,National Center for Biotechnology | Ramanculov E.,National Center for Biotechnology | Revzin A.,University of California at Davis
Analytical Chemistry | Year: 2010

In this paper, we describe the development of an electrochemical DNA aptamer-based biosensor for detection of interferon (IFN)-γ. A DNA hairpin containing IFN-γ-binding aptamer was thiolated, conjugated with methylene blue (MB) redox tag, and immobilized on a gold electrode by self-assembly. Binding of IFN-γ caused the aptamer hairpin to unfold, pushing MB redox molecules away from the electrode and decreasing electron-transfer efficiency. The change in redox current was quantified using square wave voltammetry (SWV) and was found to be highly sensitive to IFN-γ concentration. The limit of detection for optimized biosensor was 0.06 nM with linear response extending to 10 nM. This aptasensor was specific to IFN-γ in the presence of overabundant serum proteins. Importantly, the same aptasensor could be regenerated by disrupting aptamer-IFN-γ complex in urea buffer and reused multiple times. Unlike standard sandwich immunoassays, the aptasensor described here allowed one to detect IFN-γ binding directly without the need for multiple washing steps and reagents. An electrochemical biosensor for simple and sensitive detection of IFN-γ demonstrated in this paper will have future applications in immunology, cancer research, and infectious disease monitoring. © 2010 American Chemical Society.


Jones C.N.,University of California at Davis | Tuleuova N.,University of California at Davis | Tuleuova N.,National Center for Biotechnology | Lee J.Y.,University of California at Davis | And 4 more authors.
Biomaterials | Year: 2010

The goal of the present study was to investigate hepato-protective effects of growth factor (GF) arrays during alcohol injury. Hepatocyte growth factor (HGF) and bone morphogenetic protein (BMP)7 were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 500 μm diameter spots. Primary rat hepatocytes were seeded on top of the arrays forming clusters corresponding in size to the underlying protein spots. Cell arrays were then injured in culture by exposure to 100. m. m ethanol for 48. h. Hepatocytes residing on GF spots were found to have less apoptosis then cells cultured on collagen-only spots. Least apoptosis (0.3% as estimated by TUNEL assay) was observed on HGF/BMP7/collagen spots whereas most apoptosis (17.3%) was seen on collagen-only arrays. Interestingly, the extent of alcohol-induced apoptosis in hepatocytes varied based on the concentration of printed GF. In addition to preventing apoptosis, printed GFs contributed to maintenance of epithelial phenotype during alcohol injury as evidenced by higher levels of E-cadherin expression in HGF-protected hepatocytes. Importantly, GF microarrays could be used to investigate heterotypic interactions in the context of liver injury. To highlight this, stellate cells - nonparenchymal liver cells involved in fibrosis - were added to hepatocytes residing on arrays of either HGF/collagen or collagen-only spots. Exposure of these cocultures to ethanol followed by RT-PCR analysis revealed that stellate cells residing alongside HGF-protected hepatocytes were significantly less activated (less fibrotic) compared to controls. Overall, our results demonstrate that GF microarray format can be used to screen anti-fibrotic and anti-apoptotic effects of growth factors as well as to investigate how signals delivered to a specific cell type modulate heterotypic cellular interactions. © 2010 Elsevier Ltd.


Pazos F.,Computational Systems Biology Group | Pietrosemoli N.,Computational Systems Biology Group | Garcia-Martin J.A.,National Center for Biotechnology | Garcia-Martin J.A.,Boston College | Solano R.,National Center for Biotechnology
Frontiers in Plant Science | Year: 2013

To some extent contradicting the classical paradigm of the relationship between protein 3D structure and function, now it is clear that large portions of the proteomes, especially in higher organisms, lack a fixed structure and still perform very important functions. Proteins completely or partially unstructured in their native (functional) form are involved in key cellular processes underlain by complex networks of protein interactions. The intrinsic conformational flexibility of these disordered proteins allows them to bind multiple partners in transient interactions of high specificity and low affinity. In concordance, in plants this type of proteins has been found in processes requiring these complex and versatile interaction networks. These include transcription factor networks, where disordered proteins act as integrators of different signals or link different transcription factor subnetworks due to their ability to interact (in many cases simultaneously) with different partners. Similarly, they also serve as signal integrators in signaling cascades, such as those related to response to external stimuli. Disordered proteins have also been found in plants in many stress-response processes, acting as protein chaperones or protecting other cellular components and structures. In plants, it is especially important to have complex and versatile networks able to quickly and efficiently respond to changing environmental conditions since these organisms cannot escape and have no other choice than adapting to them. Consequently, protein disorder can play an especially important role in plants, providing them with a fast mechanism to obtain complex, interconnected and versatile molecular networks. © 2013 Pazos, Pietrosemoli, García-Martín and Solano.


Tuleuova N.,University of California at Davis | Tuleuova N.,National Center for Biotechnology | Jones C.N.,University of California at Davis | Yan J.,University of California at Davis | And 3 more authors.
Analytical Chemistry | Year: 2010

Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)based detection of interferon-gamma (IFN-γ)-an important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3' end resulted in the best binding IFN-γ (Kd = 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-γ binding with an aptamer beacon occurred within 15 min of analyte introduction-suggesting dynamic replacement of the quencher-complementary strand by IFN-γ molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-γ. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps, The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-γ. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokine-IFNy. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells. © 2010 American Chemical Society.


Tuleuova N.,University of California at Davis | Tuleuova N.,National Center for Biotechnology | Lee J.Y.,University of California at Davis | Lee J.,University of California at Davis | And 3 more authors.
Biomaterials | Year: 2010

The success in driving embryonic stem cells towards hepatic lineage has been confounded by the complexity and cost of differentiation protocols that employ large quantities of expensive growth factors (GFs). Instead of supplementing culture media with soluble GFs, we investigated cultivation and differentiation of mouse embryonic stem cells (mESCs) on printed arrays of GFs. Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP4) were mixed in solution with fibronectin and collagen (I) and then printed onto silane-modified glass slides to form 500 μm diameter protein spots. mESCs were cultured on top of GF spots for up to 12 days and analyzed by RT-PCR and immunostaining at different time points. The stem cells residing on HGF-containing combinations of GFs exhibited requisite features of hepatic differentiation including pronounced loss in pluripotency (Oct4), transient (up and down) expression of endoderm (Sox17) and upregulation of early hepatic markers - albumin and alpha-fetoprotein. The hepatic differentiation was enhanced further by adding hepatic stellate cells to surfaces that already contained mESCs on GF spots. A combination of co-culture with non-parenchymal liver cells and the optimal GF stimulation was found to induce endoderm and hepatic phenotype earlier and to a much greater extent than the GF arrays or micropatterned co-cultures used individually. While this paper investigated hepatic differentiation of mouse ESCs, our findings and stem cell culture approaches are likely to be relevant for human ESC cultivation. Overall, the platform combining printed GF arrays and heterotypic co-cultures will be broadly applicable for identifying the composition of the microenvironment niche for ESC differentiation into various tissue types. © 2010 Elsevier Ltd.


Golan G.,Hebrew University of Jerusalem | Ishchenko A.A.,University Paris - Sud | Khassenov B.,National Center for Biotechnology | Shoham G.,Hebrew University of Jerusalem | Saparbaev M.K.,University Paris - Sud
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis | Year: 2010

Aerobic respiration generates reactive oxygen species (ROS) as a by-product of cellular metabolism which can damage DNA. The complex nature of oxidative DNA damage requires actions of several repair pathways. Oxidized DNA bases are substrates for two overlapping pathways: base excision repair (BER) and nucleotide incision repair (NIR). In the BER pathway a DNA glycosylase cleaves the N-glycosylic bond between the abnormal base and deoxyribose, leaving either an abasic site or single-stranded DNA break. Alternatively, in the NIR pathway, an apurinic/apyrimidinic (AP) endonuclease incises duplex DNA 5′ next to oxidatively damaged nucleotide. The multifunctional Escherichia coli endonuclease IV (Nfo) is involved in both BER and NIR pathways. Nfo incises duplex DNA 5′ of a damaged residue but also possesses an intrinsic 3′ → 5′ exonuclease activity. Herein, we demonstrate that Nfo-catalyzed NIR and exonuclease activities can generate a single-strand gap at the 5′ side of 5,6-dihydrouracil residue. Furthermore, we show that Nfo mutants carrying amino acid substitutions H69A and G149D are deficient in both NIR and exonuclease activities, suggesting that these two functions are genetically linked and governed by the same amino acid residues. The crystal structure of Nfo-H69A mutant reveals the loss of one of the active site zinc atoms (Zn1) and rearrangements of the catalytic site, but no gross changes in the overall enzyme conformation. We hypothesize that these minor changes strongly affect the DNA binding of Nfo. Decreased affinity may lead to a different kinking angle of the DNA helix and this in turn thwart nucleotide incision and exonuclease activities of Nfo mutants but to lesser extent of their AP endonuclease function. Based on the biochemical and genetic data we propose a model where nucleotide incision coupled to 3′ → 5′ exonuclease activity prevents formation of lethal double-strand breaks when repairing bi-stranded clustered DNA damage. © 2009 Elsevier B.V. All rights reserved.


Mace-Aime G.,University Paris - Sud | Couve S.,University Paris - Sud | Khassenov B.,National Center for Biotechnology | Rosselli F.,University Paris - Sud | Saparbaev M.K.,University Paris - Sud
Environmental and Molecular Mutagenesis | Year: 2010

Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow failure and hypersensitivity to DNA interstrand crosslinks (ICLs) and, to a milder extension, to ionizing radiation and oxidative stress. Recently, we reported that human oxidative DNA glycosylase, NEIL1 excises with high efficiency the unhooked crosslinked oligomer within three-stranded DNA repair intermediate induced by photoactivated psoralen exposure. Complete reconstitution of repair of the ICL within a three-stranded DNA structure shows that it is processed in the short-patch base excision repair (BER) pathway. To examine whether the DNA damage hypersensitivity in FA cells follows impaired BER activities, we measured DNA glycosylase and AP endonuclease activities in cell-free extracts from wild-type, FA, and FA-corrected cells. We showed that immortalized lymphoid cells of FA complementation Groups A, C, and D and from control cells from normal donors contain similar BER activities. Intriguingly, the cellular level of NEIL1 protein strongly depends on the intact FA pathway suggesting that the hypersensitivity of FA cells to ICLs may, at least in part, arise from downregulation or degradation of NEIL1. Consistent with this result, plasmid-based expression of the FLAG-tagged NEIL1 protein partially complements the hypersensitivity FA cells to the crosslinking agents exposures, suggesting that NEIL1 specifically complements impaired capability of FA cells to repair ICLs and oxidative DNA damage. These findings shed light to how the FA pathway may regulate DNA repair proteins and bring explanation for the long-time disputed problem of the oxidative stress sensitive phenotype of FA cells. © 2010 Wiley-Liss, Inc.


News Article | November 17, 2016
Site: marketersmedia.com

— Learn Bones, one of the web’s premier resources for anatomy students and those looking to learn more about the human musculoskeletal system, recently announced the publication of their new bone health guide. The site aims to help their readers understand the underlying causes of osteoporosis and learn about the steps they can take to keep this debilitating condition at bay. The newly-published guide is currently available on the home page of the Learn Bones website at http://www.learnbones.com/. “According to the National Center for Biotechnology information, nearly 50 million U.S. citizens aged 50 and over currently suffer from osteoporosis or have bone density problems that could lead to the disease. Unfortunately, having an aging population means the problem is only expected to get worse,” said James Thompson of Learn Bones. “This is why we’ve developed and released our newest bone health guide. We want people to know that there are steps they can take to reduce their risk of developing osteoporosis and other bone conditions and give them the tools to live long, healthy, active lives,” Thompson went on to say. The recently-published bone health guide at http://www.learnbones.com/ begins with an in-depth explanation of osteoporosis and its underlying causes. The guide then goes on to explain what steps people can take right now to reduce their risk of developing it, including getting regular exercise and ensuring that they get adequate doses of daily vitamins. For those who have already developed weakness in their bones, Learn Bones’ new guide will give them tips on what to expect when it comes to possible treatments for their condition. As Thompson continued, “While everyone wants to believe that they’ll never be negatively affected by osteoporosis, the truth is that keeping our bones strong over the long haul requires us to start treating our bodies well right now. We just want to help people take control of their bone health so that they can enjoy their lives for many more years to come.” Those who would like to get a glimpse of Learn Bones’ newest bone health guide or see how they help both anatomy students and the general population better understand the human body can visit http://www.learnbones.com/ for more information. Learnbones.com is a premier resource for students of anatomy. The site offers relevant information on the bones of the human body in a concise and pertinent manner. Many of the images displayed are from Gray's Anatomy, the ultimate text book for students. In addition to information about the human skeletal system, the site also offers resources for those who want to learn about bone diseases and the muscles of the human body. For more information, please visit http://www.learnbones.com


Fernandez A.F.,University of Oviedo | Fraga M.F.,University of Oviedo | Fraga M.F.,National Center for Biotechnology
Epigenetics | Year: 2011

The physiological effects of the dietary polyphenol resveratrol are being extensively studied. Resveratrol has been proposed to promote healthy aging and to increase lifespan, primarily through the activation of the class III histone deacetylases (sirtuins). Although its positive effects are evident in yeast and mice, they still have to be confirmed in humans. The molecular mechanisms involved in the processes are not fully understood because resveratrol may have other targets than sirtuins and the direct activation of sirtuins by resveratrol is under debate. © 2011 Landes Bioscience.

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