DNA tetrahedral scaffolds-based platform for the construction of electrochemiluminescence biosensor.

PubMed

Feng, Qiu-Mei; Zhou, Zhen; Li, Mei-Xing; Zhao, Wei; Xu, Jing-Juan; Chen, Hong-Yuan

2017-04-15

Proximal metallic nanoparticles (NPs) could quench the electrochemiluminescence (ECL) emission of semiconductor quantum dots (QDs) due to Förster energy transfer (FRET), but at a certain distance, the coupling of light-emission with surface plasmon resonance (SPR) result in enhanced ECL. Thus, the modification strategies and distances control between QDs and metallic NPs are critical for the ECL intensity of QDs. In this strategy, a SPR enhanced ECL sensor based on DNA tetrahedral scaffolds modified platform was reported for the detection of telomerase activity. Due to the rigid three-dimensional structure, DNA tetrahedral scaffolds grafting on the electrode surface could accurately modulate the distance between CdS QDs and luminol labelled gold nanoparticles (L-Au NPs), meanwhile provide an enhanced spatial dimension and accessibility for the assembly of multiple L-Au NPs. The ECL intensities of both CdS QDs (-1.25V vs. SCE) and luminol (+0.33V vs. SCE) gradually increased along with the formation of multiple L-Au NPs at the vertex of DNA tetrahedral scaffolds induced by telomerase, bringing in a dual-potential ECL analysis. The proposed method showed high sensitivity for the identification of telomerase and was successfully applied for the differentiation of cancer cells from normal cells. This work suggests that DNA tetrahedral scaffolds could serve as an excellent choice for the construction of SPR-ECL system. Copyright © 2016 Elsevier B.V. All rights reserved.

Periodic Assembly of Nanospecies on Repetitive DNA Sequences Generated on Gold Nanoparticles by Rolling Circle Amplification

NASA Astrophysics Data System (ADS)

Zhao, Weian; Brook, Michael A.; Li, Yingfu

Periodical assembly of nanospecies is desirable for the construction of nanodevices. We provide a protocol for the preparation of a gold nanoparticle (AuNP)/DNA scaffold on which nanospecies can be assembled in a periodical manner. AuNP/DNA scaffold is prepared by growing long single-stranded DNA (ssDNA) molecules (typically hundreds of nanometers to a few microns in length) on AuNPs via rolling circle amplification (RCA). Since these long ssDNA molecules contain many repetitive sequence units, complementary DNA-attached nanospecies can be assembled through specific hybridization in a controllable and periodical manner.

pH-Driven Reversible Self-Assembly of Micron-Scale DNA Scaffolds.

PubMed

Green, Leopold N; Amodio, Alessia; Subramanian, Hari K K; Ricci, Francesco; Franco, Elisa

2017-12-13

Inspired by cytoskeletal scaffolds that sense and respond dynamically to environmental changes and chemical inputs with a unique capacity for reconfiguration, we propose a strategy that allows the dynamic and reversible control of the growth and breakage of micron-scale synthetic DNA structures upon pH changes. We do so by rationally designing a pH-responsive system composed of synthetic DNA strands that act as pH sensors, regulators, and structural elements. Sensor strands can dynamically respond to pH changes and route regulatory strands to direct the self-assembly of structural elements into tubular structures. This example represents the first demonstration of the reversible assembly and disassembly of micron-scale DNA scaffolds using an external chemical input other than DNA. The capacity to reversibly modulate nanostructure size may promote the development of smart devices for catalysis or drug-delivery applications.

Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification.

PubMed

Lech, Christopher Jacques; Phan, Anh Tuân

2017-06-20

Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0-13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin-avidin conjugation approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices

PubMed Central

Yan, Hao; LaBean, Thomas H.; Feng, Liping; Reif, John H.

2003-01-01

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping. PMID:12821776

Dynamic DNA-controlled "stop-and-go" assembly of well-defined protein domains on RNA-scaffolded TMV-like nanotubes.

PubMed

Schneider, Angela; Eber, Fabian J; Wenz, Nana L; Altintoprak, Klara; Jeske, Holger; Eiben, Sabine; Wege, Christina

2016-12-01

A DNA-based approach allows external control over the self-assembly process of tobacco mosaic virus (TMV)-like ribonucleoprotein nanotubes: their growth from viral coat protein (CP) subunits on five distinct RNA scaffolds containing the TMV origin of assembly (OAs) could be temporarily blocked by a stopper DNA oligomer hybridized downstream (3') of the OAs. At two upstream (5') sites tested, simple hybridization was not sufficient for stable stalling, which correlates with previous findings on a non-symmetric assembly of TMV. The growth of DNA-arrested particles could be restarted efficiently by displacement of the stopper via its toehold by using a release DNA oligomer, even after storage for twelve days. This novel strategy for growing proteinaceous tubes under tight kinetic and spatial control combines RNA guidance and its site-specific but reversible interruption by DNA blocking elements. As three of the RNA scaffolds contained long heterologous non-TMV sequence portions that included the stopping sites, this method is applicable to all RNAs amenable to TMV CP encapsidation, albeit with variable efficiency most likely depending on the scaffolds' secondary structures. The use of two distinct, selectively addressable CP variants during the serial assembly stages finally enabled an externally configured fabrication of nanotubes with highly defined subdomains. The "stop-and-go" strategy thus might pave the way towards production routines of TMV-like particles with variable aspect ratios from a single RNA scaffold, and of nanotubes with two or even more adjacent protein domains of tightly pre-defined lengths.

Folding DNA into a Lipid-Conjugated Nanobarrel for Controlled Reconstitution of Membrane Proteins.

PubMed

Dong, Yuanchen; Chen, Shuobing; Zhang, Shijian; Sodroski, Joseph; Yang, Zhongqiang; Liu, Dongsheng; Mao, Youdong

2018-02-19

Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self-assembled DNA nanobarrel that scaffolds a nanodisc-like lipid environment. Compared with the membrane-scaffolding-protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nanobarrels in the structural analysis of membrane proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scaffolded DNA origami of a DNA tetrahedron molecular container.

PubMed

Ke, Yonggang; Sharma, Jaswinder; Liu, Minghui; Jahn, Kasper; Liu, Yan; Yan, Hao

2009-06-01

We describe a strategy of scaffolded DNA origami to design and construct 3D molecular cages of tetrahedron geometry with inside volume closed by triangular faces. Each edge of the triangular face is approximately 54 nm in dimension. The estimated total external volume and the internal cavity of the triangular pyramid are about 1.8 x 10(-23) and 1.5 x 10(-23) m(3), respectively. Correct formation of the tetrahedron DNA cage was verified by gel electrophoresis, atomic force microscopy, transmission electron microscopy, and dynamic light scattering techniques.

A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

PubMed Central

Godonoga, Maia; Lin, Ting-Yu; Oshima, Azusa; Sumitomo, Koji; Tang, Marco S. L.; Cheung, Yee-Wai; Kinghorn, Andrew B.; Dirkzwager, Roderick M.; Zhou, Cunshan; Kuzuya, Akinori; Tanner, Julian A.; Heddle, Jonathan G.

2016-01-01

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology. PMID:26891622

Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade

NASA Astrophysics Data System (ADS)

Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry

2016-12-01

A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model.

PubMed

Keeney, Michael; Chung, Michael T; Zielins, Elizabeth R; Paik, Kevin J; McArdle, Adrian; Morrison, Shane D; Ransom, Ryan C; Barbhaiya, Namrata; Atashroo, David; Jacobson, Gunilla; Zare, Richard N; Longaker, Michael T; Wan, Derrick C; Yang, Fan

2016-08-01

Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016. © 2016 Wiley Periodicals, Inc.

The microviridae: Diversity, assembly, and experimental evolution.

PubMed

Doore, Sarah M; Fane, Bentley A

2016-04-01

The Microviridae, comprised of ssDNA, icosahedral bacteriophages, are a model system for studying morphogenesis and the evolution of assembly. Historically limited to the φX174-like viruses, recent results demonstrate that this richly diverse family is broadly divided into two groups. The defining feature appears to be whether one or two scaffolding proteins are required for assembly. The single-scaffolding systems contain an internal scaffolding protein, similar to many dsDNA viruses, and have a more complex coat protein fold. The two-scaffolding protein systems (φX174-like) encode an internal and external species, as well as an additional structural protein: a spike on the icosahedral vertices. Here, we discuss recent in silico and in vivo evolutionary analyses conducted with chimeric viruses and/or chimeric proteins. The results suggest 1) how double scaffolding systems can evolve into single and triple scaffolding systems; and 2) how assembly is the critical factor governing adaptation and the maintenance of species boundaries. Copyright © 2016 Elsevier Inc. All rights reserved.

Atomically precise arrays of fluorescent silver clusters: a modular approach for metal cluster photonics on DNA nanostructures.

PubMed

Copp, Stacy M; Schultz, Danielle E; Swasey, Steven; Gwinn, Elisabeth G

2015-03-24

The remarkable precision that DNA scaffolds provide for arraying nanoscale optical elements enables optical phenomena that arise from interactions of metal nanoparticles, dye molecules, and quantum dots placed at nanoscale separations. However, control of ensemble optical properties has been limited by the difficulty of achieving uniform particle sizes and shapes. Ligand-stabilized metal clusters offer a route to atomically precise arrays that combine desirable attributes of both metals and molecules. Exploiting the unique advantages of the cluster regime requires techniques to realize controlled nanoscale placement of select cluster structures. Here we show that atomically monodisperse arrays of fluorescent, DNA-stabilized silver clusters can be realized on a prototypical scaffold, a DNA nanotube, with attachment sites separated by <10 nm. Cluster attachment is mediated by designed DNA linkers that enable isolation of specific clusters prior to assembly on nanotubes and preserve cluster structure and spectral purity after assembly. The modularity of this approach generalizes to silver clusters of diverse sizes and DNA scaffolds of many types. Thus, these silver cluster nano-optical elements, which themselves have colors selected by their particular DNA templating oligomer, bring unique dimensions of control and flexibility to the rapidly expanding field of nano-optics.

Programmed folding of DNA origami structures through single-molecule force control.

PubMed

Bae, Wooli; Kim, Kipom; Min, Duyoung; Ryu, Je-Kyung; Hyeon, Changbong; Yoon, Tae-Young

2014-12-03

Despite the recent development in the design of DNA origami, its folding yet relies on thermal or chemical annealing methods. We here demonstrate mechanical folding of the DNA origami structure via a pathway that has not been accessible to thermal annealing. Using magnetic tweezers, we stretch a single scaffold DNA with mechanical tension to remove its secondary structures, followed by base pairing of the stretched DNA with staple strands. When the force is subsequently quenched, folding of the DNA nanostructure is completed through displacement between the bound staple strands. Each process in the mechanical folding is well defined and free from kinetic traps, enabling us to complete folding within 10 min. We also demonstrate parallel folding of DNA nanostructures through multiplexed manipulation of the scaffold DNAs. Our results suggest a path towards programmability of the folding pathway of DNA nanostructures.

MD simulations of papillomavirus DNA-E2 protein complexes hints at a protein structural code for DNA deformation.

PubMed

Falconi, M; Oteri, F; Eliseo, T; Cicero, D O; Desideri, A

2008-08-01

The structural dynamics of the DNA binding domains of the human papillomavirus strain 16 and the bovine papillomavirus strain 1, complexed with their DNA targets, has been investigated by modeling, molecular dynamics simulations, and nuclear magnetic resonance analysis. The simulations underline different dynamical features of the protein scaffolds and a different mechanical interaction of the two proteins with DNA. The two protein structures, although very similar, show differences in the relative mobility of secondary structure elements. Protein structural analyses, principal component analysis, and geometrical and energetic DNA analyses indicate that the two transcription factors utilize a different strategy in DNA recognition and deformation. Results show that the protein indirect DNA readout is not only addressable to the DNA molecule flexibility but it is finely tuned by the mechanical and dynamical properties of the protein scaffold involved in the interaction.

Detection of Intermediates And Kinetic Control During Assembly of Bacteriophage P22 Procapsid

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

Tuma, R.; Tsuruta, H.; French, K.H.

2009-05-26

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter {approx} 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibriummore » (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.« less

Releasing the cohesin ring: A rigid scaffold model for opening the DNA exit gate by Pds5 and Wapl.

PubMed

Ouyang, Zhuqing; Yu, Hongtao

2017-04-01

The ring-shaped ATPase machine, cohesin, regulates sister chromatid cohesion, transcription, and DNA repair by topologically entrapping DNA. Here, we propose a rigid scaffold model to explain how the cohesin regulators Pds5 and Wapl release cohesin from chromosomes. Recent studies have established the Smc3-Scc1 interface as the DNA exit gate of cohesin, revealed a requirement for ATP hydrolysis in ring opening, suggested regulation of the cohesin ATPase activity by DNA and Smc3 acetylation, and provided insights into how Pds5 and Wapl open this exit gate. We hypothesize that Pds5, Wapl, and SA1/2 form a rigid scaffold that docks on Scc1 and anchors the N-terminal domain of Scc1 (Scc1N) to the Smc1 ATPase head. Relative movements between the Smc1-3 ATPase heads driven by ATP and Wapl disrupt the Smc3-Scc1 interface. Pds5 binds the dissociated Scc1N and prolongs this open state of cohesin, releasing DNA. We review the evidence supporting this model and suggest experiments that can further test its key principles. © 2017 WILEY Periodicals, Inc.

An Insilico Design of Nanoclay Based Nanocomposites and Scaffolds in Bone Tissue Engineering

NASA Astrophysics Data System (ADS)

Sharma, Anurag

A multiscale in silico approach to design polymer nanocomposites and scaffolds for bone tissue engineering applications is described in this study. This study focuses on the role of biomaterials design and selection, structural integrity and mechanical properties evolution during degradation and tissue regeneration in the successful design of polymer nanocomposite scaffolds. Polymer nanocomposite scaffolds are synthesized using aminoacid modified montmorillonite nanoclay with biomineralized hydroxyapatite and polycaprolactone (PCL/in situ HAPclay). Representative molecular models of polymer nanocomposite system are systematically developed using molecular dynamics (MD) technique and successfully validated using material characterization techniques. The constant force steered molecular dynamics (fSMD) simulation results indicate a two-phase nanomechanical behavior of the polymer nanocomposite. The MD and fSMD simulations results provide quantitative contributions of molecular interactions between different constituents of representative models and their effect on nanomechanical responses of nanoclay based polymer nanocomposite system. A finite element (FE) model of PCL/in situ HAPclay scaffold is built using micro-computed tomography images and bridging the nanomechanical properties obtained from fSMD simulations into the FE model. A new reduction factor, K is introduced into modeling results to consider the effect of wall porosity of the polymer scaffold. The effect of accelerated degradation under alkaline conditions and human osteoblast cells culture on the evolution of mechanical properties of scaffolds are studied and the damage mechanics based analytical models are developed. Finally, the novel multiscale models are developed that incorporate the complex molecular and microstructural properties, mechanical properties at nanoscale and structural levels and mechanical properties evolution during degradation and tissue formation in the polymer nanocomposite scaffold. Overall, this study provides a leap into methodologies for in silico design of biomaterials for bone tissue engineering applications. Furthermore, as a part of this work, a molecular dynamics study of rice DNA in the presence of single walled carbon nanotube is carried out to understand the role played by molecular interactions in the conformation changes of rice DNA. The simulations results showed wrapping of DNA onto SWCNT, breaking and forming of hydrogen bonds due to unzipping of Watson-Crick (WC) nucleobase pairs and forming of new non-WC nucleobase pairs in DNA.

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

PubMed Central

Khojasteh, Arash; Motamedian, Saeed Reza; Rad, Maryam Rezai; Shahriari, Mehrnoosh Hasan; Nadjmi, Nasser

2015-01-01

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells (hDPSCs) on four commercially available scaffold biomaterials. METHODS: hDPSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. hDPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureOss (Allograft), Cerabone (Xenograft), PLLA (Synthetic), and OSTEON II Collagen (Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy (SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase (ALP) activity assay, respectively. RESULTS: All scaffold biomaterials except SureOss (Allograft) supported hDPSC adhesion, proliferation and differentiation. hDPSCs seeded on PLLA (Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hDPSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA (Synthetic) and OSTEON II Collagen (Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone (Xenograft) and OSTEON II Collagen (Composite) scaffolds, the hDPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape. CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hDPSCs. Hence, it may be useful in combination with hDPSCs for cell-based reconstructive therapy. PMID:26640621

Incorporation of Active DNA/Cationic Polymer Polyplexes into Hydrogel Scaffolds

PubMed Central

Lei, Yuguo; Huang, Suxian; Sharif-Kashani, Pooria; Chen, Yong; Kavehpour, Pirouz; Segura, Tatiana

2010-01-01

The effective and sustained delivery of DNA and siRNAs locally would increase the applicability of gene therapy in tissue regeneration and cancer therapy. One promising approach is to use hydrogel scaffolds to encapsulate and deliver nucleotides in the form of nanoparticles to the disease sites. However, this approach is currently limited by the inability to load concentrated and active gene delivery nanoparticles into the hydrogels due to the severe nanoparticle aggregation during the loading process. Here, we present a process to load concentrated and un-aggregated non-viral gene delivery nanoparticles, using DNA/polyethylene imine (PEI) polyplexes as an example, into neutral polyethylene glycol (PEG), negatively charged hyaluronic acid (HA) and protein fibrin hydrogels crosslinked through various chemistries. The encapsulated polyplexes are highly active both in vitro and in vivo. We believe this process will significantly advance the applications of hydrogel scaffold mediated non-viral gene delivery in tissue regeneration and cancer therapy. PMID:20822811

Impact of pre-incubation time of silk fibroin scaffolds in culture medium on cell proliferation and attachment.

PubMed

Amirikia, Mehdi; Shariatzadeh, Seyed Mohammad Ali; Jorsaraei, Seyed Gholam Ali; Soleimani Mehranjani, Malek

2017-12-01

Cell behaviours such as proliferation and attachment can be affected by the length of pre-incubation period of the scaffolds in the culture medium for long term. The aim of this study was to investigate the long term pre-incubation of 3D silk fibroin scaffolds in complete culture medium on cell attachment and proliferation. After the preparation of silk fibroin scaffolds by the technique of freeze drying, the scaffolds were pre-incubated in complete culture medium for 2 d, 6 d or 10 d before apical papilla stem cells (SCAP) seeding. Modifications of the scaffold surface and wettability were examined by FE-SEM and water contact angle, respectively. Results showed a decrease both in roughness and water contact angle as pre-incubation time increases. DNA measurement after 18h and 10 d cell seeding showed a significant increase of DNA concentration which represents better attachment and proliferation with pre-incubation time increase. Qualitative examination, live&dead assay or H&E staining method after 30h and 10 d cell seeding respectively, indicated that pre-incubation of scaffolds has time dependent effect on cell proliferation and attachment. This suggests that improvement of cell attachment and proliferation may be mediated by differences in the amount of wettability (decreased water contact angle) after exposure of scaffold to culture medium for long term which, in turn, causes more protein adsorption in the surface of silk fibroin scaffold (decreased roughness). Copyright © 2017. Published by Elsevier Ltd.

Designing DNA nanodevices for compatibility with the immune system of higher organisms

NASA Astrophysics Data System (ADS)

Surana, Sunaina; Shenoy, Avinash R.; Krishnan, Yamuna

2015-09-01

DNA is proving to be a powerful scaffold to construct molecularly precise designer DNA devices. Recent trends reveal their ever-increasing deployment within living systems as delivery devices that not only probe but also program and re-program a cell, or even whole organisms. Given that DNA is highly immunogenic, we outline the molecular, cellular and organismal response pathways that designer nucleic acid nanodevices are likely to elicit in living systems. We address safety issues applicable when such designer DNA nanodevices interact with the immune system. In light of this, we discuss possible molecular programming strategies that could be integrated with such designer nucleic acid scaffolds to either evade or stimulate the host response with a view to optimizing and widening their applications in higher organisms.

Combining technologies to create bioactive hybrid scaffolds for bone tissue engineering.

PubMed

Nandakumar, Anandkumar; Barradas, Ana; de Boer, Jan; Moroni, Lorenzo; van Blitterswijk, Clemens; Habibovic, Pamela

2013-01-01

Combining technologies to engineer scaffolds that can offer physical and chemical cues to cells is an attractive approach in tissue engineering and regenerative medicine. In this study, we have fabricated polymer-ceramic hybrid scaffolds for bone regeneration by combining rapid prototyping (RP), electrospinning (ESP) and a biomimetic coating method in order to provide mechanical support and a physico-chemical environment mimicking both the organic and inorganic phases of bone extracellular matrix (ECM). Poly(ethylene oxide terephthalate)-poly(buthylene terephthalate) (PEOT/PBT) block copolymer was used to produce three dimensional scaffolds by combining 3D fiber (3DF) deposition, and ESP, and these constructs were then coated with a Ca-P layer in a simulated physiological solution. Scaffold morphology and composition were studied using scanning electron microscopy (SEM) coupled to energy dispersive X-ray analyzer (EDX) and Fourier Tranform Infrared Spectroscopy (FTIR). Bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on coated and uncoated 3DF and 3DF + ESP scaffolds for up to 21 d in basic and mineralization medium and cell attachment, proliferation, and expression of genes related to osteogenesis were assessed. Cells attached, proliferated and secreted ECM on all the scaffolds. There were no significant differences in metabolic activity among the different groups on days 7 and 21. Coated 3DF scaffolds showed a significantly higher DNA amount in basic medium at 21 d compared with the coated 3DF + ESP scaffolds, whereas in mineralization medium, the presence of coating in 3DF+ESP scaffolds led to a significant decrease in the amount of DNA. An effect of combining different scaffolding technologies and material types on expression of a number of osteogenic markers (cbfa1, BMP-2, OP, OC and ON) was observed, suggesting the potential use of this approach in bone tissue engineering.

Programmable self-assembly of three-dimensional nanostructures from 104 unique components

PubMed Central

Ong, Luvena L.; Hanikel, Nikita; Yaghi, Omar K.; Grun, Casey; Strauss, Maximilian T.; Bron, Patrick; Lai-Kee-Him, Josephine; Schueder, Florian; Wang, Bei; Wang, Pengfei; Kishi, Jocelyn Y.; Myhrvold, Cameron A.; Zhu, Allen; Jungmann, Ralf

2017-01-01

Nucleic acids (DNA and RNA) are widely used to construct nanoscale structures with ever increasing complexity1–14 for possible applications in fields as diverse as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early examples typically containing on the order of 10 unique DNA strands. The introduction of DNA origami4, which uses many staple strands to fold one long scaffold strand into a desired structure, gave access to kilo- to mega-dalton nanostructures containing about 102 unique DNA strands6,7,10,13 . Aiming for even larger DNA origami structures is in principle possible15,16, but faces the challenge of having to manufacture and route an increasingly long scaffold strand. An alternative and in principle more readily scalable approach uses DNA brick assembly8,9, which doesn’t need a scaffold and instead uses hundreds of short DNA brick strands that self-assemble according to specific inter-brick interactions. First-generation bricks used to create 3D structures are 32-nt long with four 8-nt binding domains that directed 102 distinct bricks into well-formed assemblies, but attempts to create larger structures encountered practical challenges and had limited success.9 Here we show that a new generation of DNA bricks with longer binding domains makes it possible to self-assemble 0.1 – 1 giga-dalton three-dimensional nanostructures from 104 unique components, including a 0.5 giga-dalton cuboid containing 30,000 unique bricks and a 1 giga-dalton rotationally symmetric tetramer. We also assemble a cuboid containing 10,000 bricks and 20,000 uniquely addressable ‘nano-voxels’ that serves as a molecular canvas for three-dimensional sculpting, with introduction of sophisticated user-prescribed 3D cavities yielding structures such as letters, a complex helicoid and a teddy bear. We anticipate that, with further optimization, even larger assemblies might be accessible and prove useful as scaffolds or for positioning functional components. PMID:29219968

The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF(165).

PubMed

Keeney, Michael; van den Beucken, Jeroen J J P; van der Kraan, Peter M; Jansen, John A; Pandit, Abhay

2010-04-01

Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherent similarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viral vector for gene delivery. The aim of this study was to determine the capability of a collagen/calcium phosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of the study was to deliver a plasmid encoding vascular endothelial growth factor(165) (pVEGF(165)) to promote angiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmid DNA resulted in a 7.6-fold increase in mRNA levels of beta-Galactosidase compared to the delivery of plasmid DNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murine model. When implanted in a muirne intra-femoral model, the delivery of pVEGF(165) resulted in a 2-fold increase in bone volume at the defect site relative to control scaffolds without pVEGF(165). It was concluded that a collagen/calcium phosphate scaffold can mediate transfection without the use of additional transfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF(165). 2009 Elsevier Ltd. All rights reserved.

Hydroxyapatite/regenerated silk fibroin scaffold-enhanced osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells.

PubMed

Jiang, Jia; Hao, Wei; Li, Yuzhuo; Yao, Jinrong; Shao, Zhengzhong; Li, Hong; Yang, Jianjun; Chen, Shiyi

2013-04-01

A novel hydroxyapatite/regenerated silk fibroin scaffold was prepared and investigated for its potential to enhance both osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells in vitro. Approx. 12.4 ± 0.06 % (w/w) hydroxyapatite was deposited onto the scaffold, and cell viability and DNA content were significantly increased (18.5 ± 0.6 and 33 ± 1.2 %, respectively) compared with the hydroxyapatite scaffold after 14 days. Furthermore, alkaline phosphatase activity in the novel scaffold increased 41 ± 2.5 % after 14 days compared with the hydroxyapatite scaffold. The data indicate that this novel hydroxyapatite/regenerated silk fibroin scaffold has a positive effect on osteoinductivity and osteoconductivity, and may be useful for bone tissue engineering.

Antiproliferative, DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: A structure-activity relationship study.

PubMed

Ho, Si-Han Sherman; Sim, Mei-Yi; Yee, Wei-Loong Sherman; Yang, Tianming; Yuen, Shyi-Peng John; Go, Mei-Lin

2015-11-02

The anticancer agent YM155 is widely investigated as a specific survivin suppressant. More recently, YM155 was found to induce DNA damage and this has raised doubts as to whether survivin is its primary target. In an effort to assess the contribution of DNA damage to the anticancer activity of YM155, several analogs were prepared and evaluated for antiproliferative activity on malignant cells, participation in DNA intercalation and free radical generation by redox cycling. The intact positively charged scaffold was found to be essential for antiproliferative activity and intercalation but was less critical for redox cycling where the minimal requirement was a pared down bicyclic quinone. Side chain requirements at the N(1) and N(3) positions of the scaffold were more alike for redox cycling and intercalation than antiproliferative activity, underscoring yet again, the limited structural overlaps for these activities. Furthermore, antiproliferative activities were poorly correlated to DNA intercalation and redox cycling. Potent antiproliferative activity (IC50 9-23 nM), exceeding that of YM155, was found for a minimally substituted methyl analog AB7. Like YM155 and other dioxonaphthoimidazoliums, AB7 was a modest DNA intercalator but with weak redox cycling activity. Thus, the capacity of this scaffold to inflict direct DNA damage leading to cell death may not be significant and YM155 should not be routinely classified as a DNA damaging agent. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

DNA-Templated Pd Conductive Metallic Nanowires

NASA Astrophysics Data System (ADS)

Nguyen, K.; Monteverde, M.; Lyonnais, S.; Campidelli, S.; Bourgoin, J.-Ph.; Filoramo, A.

2008-10-01

Because of its unique recognition properties, its size and the sub-nanometric resolution, DNA is of particular interest for positioning and organizing nanomaterials. However, in DNA-directed nanoelectronic it can be envisioned to use DNA not only as a positioning scaffold, but also as a support for the conducting element. To ensure this function a metallization process is necessary and among the various DNA metallization methods the Pd based ones are of particular interest for carbon nanotube transistor connections. In this field, the major drawback of the existing methods is the fast kinetics of the process which lead to a stochastic growth. Here, we present a novel approach to DNA Pd metalization where the DNA molecule is previously deposited on a dry substrate in a typical nanodevice configuration. In our approach the progressive growth of nanowires is achieved by the slow and selective precipitation of PdO, followed by a subsequent reduction step. Thanks to this strategy we fabricated homogeneous, continuous and conductive Pd nanowires on the DNA scaffolds of very thin diameter (20-25 nm).

Platelet-rich plasma combined with agarose as a bioactive scaffold to enhance cartilage repair: an in vitro study.

PubMed

Yin, Zhaowei; Yang, Xiaofei; Jiang, Yiqiu; Xing, Linzi; Xu, Yang; Lu, Yiming; Ding, Peng; Ma, Junxin; Xu, Yan; Gui, Jianchao

2014-03-01

The purpose of this study was to determine whether the platelet-rich plasma-agarose gel scaffold could be a bioactive scaffold capable of growth factors release for cartilage repair. Porcine chondrocytes were seeded in agarose gel and platelet-rich plasma-agarose gel. During the 28-days culture, microstructure of hydrogels and morphologies of chondrocytes seeded in the hydrogels were observed using scanning electron microscope; viability of chondrocytes in gels was examined by live/dead assay; qualitative and quantitative analysis of glycosaminoglycan, collagen and DNA were assessed by histological, immunohistochemical staining and biochemical assay; gene expression was measured by real-time polymerase chain reaction. In vitro cartilage ring models were used to evaluate the integration of the scaffolds, and the integration strength was analyzed by mechanical push-out tests. Scanning electron microscope revealed both scaffolds had highly uniform porous structure. Live/dead scaffolds showed 100% cells alive in both groups. After 28-days culture, glycosaminoglycan, collagen, DNA content and chondrocyte-related genes expression in platelet-rich plasma-agarose gel were significantly higher than pure agarose gel. Integration strength in platelet-rich plasma-agarose gel was also higher compared to pure agarose gel. Platelet-rich plasma showed a positive effect on chondrocytes proliferation, differentiation and integration between native cartilage and engineered tissue when combined with agarose gel. Our findings suggest that platelet-rich plasma-agarose gel scaffold is a promising bioactive scaffold for future cartilage tissue engineering and future clinical works.

Self-assembled DNA Structures for Nanoconstruction

NASA Astrophysics Data System (ADS)

Yan, Hao; Yin, Peng; Park, Sung Ha; Li, Hanying; Feng, Liping; Guan, Xiaoju; Liu, Dage; Reif, John H.; LaBean, Thomas H.

2004-09-01

In recent years, a number of research groups have begun developing nanofabrication methods based on DNA self-assembly. Here we review our recent experimental progress to utilize novel DNA nanostructures for self-assembly as well as for templates in the fabrication of functional nano-patterned materials. We have prototyped a new DNA nanostructure known as a cross structure. This nanostructure has a 4-fold symmetry which promotes its self-assembly into tetragonal 2D lattices. We have utilized the tetragonal 2D lattices as templates for highly conductive metallic nanowires and periodic 2D protein nano-arrays. We have constructed and characterized a DNA nanotube, a new self-assembling superstructure composed of DNA tiles. We have also demonstrated an aperiodic DNA lattice composed of DNA tiles assembled around a long scaffold strand; the system translates information encoded in the scaffold strand into a specific and reprogrammable barcode pattern. We have achieved metallic nanoparticle linear arrays templated on self-assembled 1D DNA arrays. We have designed and demonstrated a 2-state DNA lattice, which displays expand/contract motion switched by DNA nanoactuators. We have also achieved an autonomous DNA motor executing unidirectional motion along a linear DNA track.

M1.3 - a small scaffold for DNA origami

NASA Astrophysics Data System (ADS)

Said, Hassan; Schüller, Verena J.; Eber, Fabian J.; Wege, Christina; Liedl, Tim; Richert, Clemens

2012-12-01

The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed ``M1.3'' as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments.The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed ``M1.3'' as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments. Electronic supplementary information (ESI) available: Materials, full sequence of M1.3, alternative restriction reactions, sequences and origami designs, ALEX data, estimated cost of producing M13, additional melting data for origami, and MALDI spectra of individual capped oligonucleotides. See DOI: 10.1039/c2nr32393a

Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production.

PubMed

Kick, Benjamin; Hensler, Samantha; Praetorius, Florian; Dietz, Hendrik; Weuster-Botz, Dirk

2017-04-01

The bacteriophage M13 has found frequent applications in nanobiotechnology due to its chemically and genetically tunable protein surface and its ability to self-assemble into colloidal membranes. Additionally, its single-stranded (ss) genome is commonly used as scaffold for DNA origami. Despite the manifold uses of M13, upstream production methods for phage and scaffold ssDNA are underexamined with respect to future industrial usage. Here, the high-cell-density phage production with Escherichia coli as host organism was studied in respect of medium composition, infection time, multiplicity of infection, and specific growth rate. The specific growth rate and the multiplicity of infection were identified as the crucial state variables that influence phage amplification rate on one hand and the concentration of produced ssDNA on the other hand. Using a growth rate of 0.15 h -1 and a multiplicity of infection of 0.05 pfu cfu -1 in the fed-batch production process, the concentration of pure isolated M13 ssDNA usable for scaffolded DNA origami could be enhanced by 54% to 590 mg L -1 . Thus, our results help enabling M13 production for industrial uses in nanobiotechnology. Biotechnol. Bioeng. 2017;114: 777-784. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Engineering DNA Backbone Interactions Results in TALE Scaffolds with Enhanced 5-Methylcytosine Selectivity.

PubMed

Rathi, Preeti; Witte, Anna; Summerer, Daniel

2017-11-08

Transcription activator-like effectors (TALEs) are DNA major-groove binding proteins widely used for genome targeting. TALEs contain an N-terminal region (NTR) and a central repeat domain (CRD). Repeats of the CRD selectively recognize each one DNA nucleobase, offering programmability. Moreover, repeats with selectivity for 5-methylcytosine (5mC) and its oxidized derivatives can be designed for analytical applications. However, both TALE domains also nonspecifically interact with DNA phosphates via basic amino acids. To enhance the 5mC selectivity of TALEs, we aimed to decrease the nonselective binding energy of TALEs. We substituted basic amino acids with alanine in the NTR and identified TALE mutants with increased selectivity. We then analysed conserved, DNA phosphate-binding KQ diresidues in CRD repeats and identified further improved mutants. Combination of mutations in the NTR and CRD was highly synergetic and resulted in TALE scaffolds with up to 4.3-fold increased selectivity in genomic 5mC analysis via affinity enrichment. Moreover, transcriptional activation in HEK293T cells by a TALE-VP64 construct based on this scaffold design exhibited a 3.5-fold increased 5mC selectivity. This provides perspectives for improved 5mC analysis and for the 5mC-conditional control of TALE-based editing constructs in vivo.

Application of DNA Machineries for the Barcode Patterned Detection of Genes or Proteins.

PubMed

Zhou, Zhixin; Luo, Guofeng; Wulf, Verena; Willner, Itamar

2018-06-05

The study introduces an analytical platform for the detection of genes or aptamer-ligand complexes by nucleic acid barcode patterns generated by DNA machineries. The DNA machineries consist of nucleic acid scaffolds that include specific recognition sites for the different genes or aptamer-ligand analytes. The binding of the analytes to the scaffolds initiate, in the presence of the nucleotide mixture, a cyclic polymerization/nicking machinery that yields displaced strands of variable lengths. The electrophoretic separation of the resulting strands provides barcode patterns for the specific detection of the different analytes. Mixtures of DNA machineries that yield, upon sensing of different genes (or aptamer ligands), one-, two-, or three-band barcode patterns are described. The combination of nucleic acid scaffolds acting, in the presence of polymerase/nicking enzyme and nucleotide mixture, as DNA machineries, that generate multiband barcode patterns provide an analytical platform for the detection of an individual gene out of many possible genes. The diversity of genes (or other analytes) that can be analyzed by the DNA machineries and the barcode patterned imaging is given by the Pascal's triangle. As a proof-of-concept, the detection of one of six genes, that is, TP53, Werner syndrome, Tay-Sachs normal gene, BRCA1, Tay-Sachs mutant gene, and cystic fibrosis disorder gene by six two-band barcode patterns is demonstrated. The advantages and limitations of the detection of analytes by polymerase/nicking DNA machineries that yield barcode patterns as imaging readout signals are discussed.

C 3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

PubMed Central

McStay, Natasha; Molphy, Zara; Coughlan, Alan; Cafolla, Attilio; McKee, Vickie; Gathergood, Nicholas; Kellett, Andrew

2017-01-01

Herein we report the synthesis of tripodal C3-symmetric opioid scaffolds as high-affinity condensation agents of duplex DNA. Condensation was achieved on both supercoiled and canonical B-DNA structures and identified by agarose electrophoresis, viscosity, turbidity and atomic force microscopy (AFM) measurements. Structurally, the requirement of a tris-opioid scaffold for condensation is demonstrated as both di- (C2-symmetric) and mono-substituted (C1-symmetric) mesitylene-linked opioid derivatives poorly coordinate dsDNA. Condensation, observed by toroidal and globule AFM aggregation, arises from surface-binding ionic interactions between protonated, cationic, tertiary amine groups on the opioid skeleton and the phosphate nucleic acid backbone. Indeed, by converting the 6-hydroxyl group of C3-morphine (MC3) to methoxy substituents in C3-heterocodeine (HC3) and C3-oripavine (OC3) molecules, dsDNA compaction is retained thus negating the possibility of phosphate—hydroxyl surface-binding. Tripodal opioid condensation was identified as pH dependent and strongly influenced by ionic strength with further evidence of cationic amine-phosphate backbone coordination arising from thermal melting analysis and circular dichroism spectroscopy, with compaction also witnessed on synthetic dsDNA co-polymers poly[d(A-T)2] and poly[d(G-C)2]. On-chip microfluidic analysis of DNA condensed by C3-agents provided concentration-dependent protection (inhibition) to site-selective excision by type II restriction enzymes: BamHI, HindIII, SalI and EcoRI, but not to the endonuclease DNase I. PMID:27899572

Magnesium Presence Prevents Removal of Antigenic Nuclear-Associated Proteins from Bovine Pericardium for Heart Valve Engineering.

PubMed

Dalgliesh, Ailsa J; Liu, Zhi Zhao; Griffiths, Leigh G

2017-07-01

Current heart valve prostheses are associated with significant complications, including aggressive immune response, limited valve life expectancy, and inability to grow in juvenile patients. Animal derived "tissue" valves undergo glutaraldehyde fixation to mask tissue antigenicity; however, chronic immunological responses and associated calcification still commonly occur. A heart valve formed from an unfixed bovine pericardium (BP) extracellular matrix (ECM) scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current bioprostheses. Decellularization and antigen removal methods frequently use sequential solutions extrapolated from analytical chemistry approaches to promote solubility and removal of tissue components from resultant ECM scaffolds. However, the extent to which such prefractionation strategies may inhibit removal of antigenic tissue components has not been explored. We hypothesize that presence of magnesium in prefractionation steps causes DNA precipitation and reduces removal of nuclear-associated antigenic proteins. Keeping all variables consistent bar the addition or absence of magnesium (2 mM magnesium chloride hexahydrate), residual BP ECM scaffold antigenicity and removed antigenicity were assessed, along with residual and removed DNA content, ECM morphology, scaffold composition, and recellularization potential. Furthermore, we used proteomic methods to determine the mechanism by which magnesium presence or absence affects scaffold residual antigenicity. This study demonstrates that absence of magnesium from antigen removal solutions enhances solubility and subsequent removal of antigenic nuclear-associated proteins from BP. We therefore conclude that the primary mechanism of action for magnesium removal during antigen removal processes is avoidance of DNA precipitation, facilitating solubilization and removal of nuclear-associated antigenic proteins. Future studies are necessary to further facilitate solubility and removal of nuclear-associated antigenic proteins from xenogeneic ECM scaffolds, in addition to an in vivo assessing of the material.

Design and Characterization of Micro-Porous Hyaluronic Acid Hydrogels for in vitro Gene Transfer to mMSCs

PubMed Central

Tokatlian, Talar; Cam, Cynthia; Siegman, Shayne N.; Lei, Yuguo; Segura, Tatiana

2013-01-01

The effective and sustained delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration and therapeutic angiogenesis. One promising approach is to use porous hydrogel scaffolds to encapsulate and deliver nucleotides in the form of nanoparticles to the affected sites. We have designed and characterized micro-porous (µ-pore) hyaluronic acid hydrogels which allow for effective cell seeding in vitro post scaffold fabrication and allow for cell spreading and proliferation without requiring high levels of degradation. These factors, coupled with high loading efficiency of DNA polyplexes using a previously developed caged nanoparticle encapsulation (CnE) technique, then allowed for long-term sustained transfection and transgene expression of incorporated mMSCs. In this study, we examined the effect of pore size on gene transfer efficiency and the kinetics of transgene expression. For all investigated pore sizes (30, 60, and 100 µm), encapsulated DNA polyplexes were released steadily starting by day 4 for up to 10 days. Likewise, transgene expression was sustained over this period, although significant differences between different pore sizes were not observed. Cell viability was also shown to remain high over time, even in the presence of high concentrations of DNA polyplexes. The knowledge acquired through this in vitro model can be utilized to design and better predict scaffold-mediated gene delivery for local gene therapy in an in vivo model where host cells infiltrate the scaffold over time. PMID:22820309

Polymorphic design of DNA origami structures through mechanical control of modular components.

PubMed

Lee, Chanseok; Lee, Jae Young; Kim, Do-Nyun

2017-12-12

Scaffolded DNA origami enables the bottom-up fabrication of diverse DNA nanostructures by designing hundreds of staple strands, comprised of complementary sequences to the specific binding locations of a scaffold strand. Despite its exceptionally high design flexibility, poor reusability of staples has been one of the major hurdles to fabricate assorted DNA constructs in an effective way. Here we provide a rational module-based design approach to create distinct bent shapes with controllable geometries and flexibilities from a single, reference set of staples. By revising the staple connectivity within the desired module, we can control the location, stiffness, and included angle of hinges precisely, enabling the construction of dozens of single- or multiple-hinge structures with the replacement of staple strands up to 12.8% only. Our design approach, combined with computational shape prediction and analysis, can provide a versatile and cost-effective procedure in the design of DNA origami shapes with stiffness-tunable units.

Comprehensive Genetic Analysis of Early Host Body Reactions to the Bioactive and Bio-Inert Porous Scaffolds

PubMed Central

Ehashi, Tomo; Takemura, Taro; Hanagata, Nobutaka; Minowa, Takashi; Kobayashi, Hisatoshi; Ishihara, Kazuhiko; Yamaoka, Tetsuji

2014-01-01

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1β and IL-10 are important cytokines in tissue responses to biomaterials because IL-1β promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1β was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations. PMID:24454803

Comparative scaffolding and gap filling of ancient bacterial genomes applied to two ancient Yersinia pestis genomes

PubMed Central

Doerr, Daniel; Chauve, Cedric

2017-01-01

Yersinia pestis is the causative agent of the bubonic plague, a disease responsible for several dramatic historical pandemics. Progress in ancient DNA (aDNA) sequencing rendered possible the sequencing of whole genomes of important human pathogens, including the ancient Y. pestis strains responsible for outbreaks of the bubonic plague in London in the 14th century and in Marseille in the 18th century, among others. However, aDNA sequencing data are still characterized by short reads and non-uniform coverage, so assembling ancient pathogen genomes remains challenging and often prevents a detailed study of genome rearrangements. It has recently been shown that comparative scaffolding approaches can improve the assembly of ancient Y. pestis genomes at a chromosome level. In the present work, we address the last step of genome assembly, the gap-filling stage. We describe an optimization-based method AGapEs (ancestral gap estimation) to fill in inter-contig gaps using a combination of a template obtained from related extant genomes and aDNA reads. We show how this approach can be used to refine comparative scaffolding by selecting contig adjacencies supported by a mix of unassembled aDNA reads and comparative signal. We applied our method to two Y. pestis data sets from the London and Marseilles outbreaks, for which we obtained highly improved genome assemblies for both genomes, comprised of, respectively, five and six scaffolds with 95 % of the assemblies supported by ancient reads. We analysed the genome evolution between both ancient genomes in terms of genome rearrangements, and observed a high level of synteny conservation between these strains. PMID:29114402

Orthogonal labeling of M13 minor capsid proteins with DNA to self-assemble end-to-end multiphage structures.

PubMed

Hess, Gaelen T; Guimaraes, Carla P; Spooner, Eric; Ploegh, Hidde L; Belcher, Angela M

2013-09-20

M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.

Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

PubMed Central

Guo, Fei; Liu, Zheng; Fang, Ping-An; Zhang, Qinfen; Wright, Elena T.; Wu, Weimin; Zhang, Ci; Vago, Frank; Ren, Yue; Jakana, Joanita; Chiu, Wah; Serwer, Philip; Jiang, Wen

2014-01-01

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses. PMID:25313071

A DNA-Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition.

PubMed

Favalli, Nicholas; Biendl, Stefan; Hartmann, Marco; Piazzi, Jacopo; Sladojevich, Filippo; Gräslund, Susanne; Brown, Peter J; Näreoja, Katja; Schüler, Herwig; Scheuermann, Jörg; Franzini, Raphael; Neri, Dario

2018-06-01

A DNA-encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5-bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a K d value of 6 nm, while compounds with the same substituents on an equidistant but flexible l-lysine scaffold showed 140-fold lower affinity. A 18 nm tankyrase-1 binder featured l-lysine as linking moiety, while molecules based on d-Lysine or (2S,4S)-amino-l-proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhancement of Cell Ingrowth, Proliferation, and Early Differentiation in a Three-Dimensional Silicon Carbide Scaffold Using Low-Intensity Pulsed Ultrasound

PubMed Central

Lin, Liangjun

2015-01-01

Concerns over the use of autografts or allografts have necessitated the development of biomaterials for bone regeneration. Various studies have been performed to optimize the cultivation of osteogenic cells using osteoconductive porous scaffolds. The aim of this study was to evaluate the osteogenic efficiency of bone cell ingrowth, proliferation, and early differentiation in a silicon carbide (SiC) porous ceramic scaffold promoted with low-intensity pulsed ultrasound. MC3T3-E1 mouse preosteoblasts were seeded onto scaffolds and cultured for 4 and 7 days with daily of 20-min ultrasound treatment. The cells were evaluated for cell attachment, morphology, viability, ingrowth depth, volumetric proliferation, and early differentiation. After 4 and 7 days of culture and ultrasound exposure, the cell density was higher in the ultrasound-treated group compared with the sham-treated group on SiC scaffolds. The cell ingrowth depths inside the SiC scaffolds were 149.2±27.3 μm at 1 day, 310.1±12.6 μm for the ultrasound-treated group and 248.0±19.7 μm for the sham control at 4 days, and 359.6±18.5 μm for the ultrasound-treated group and 280.0±17.7 μm for the sham control at 7 days. They were significantly increased, that is, 25% (p=0.0029) and 28% (p=0.0008) increase, respectively, with ultrasound radiation force as compared with those in sham control at 4 and 7 days postseeding. The dsDNA contents were 583.5±19.1 ng/scaffold at 1 day, 2749.9±99.9 ng/scaffold for the ultrasound-treated group and 2514.9±114.7 ng/scaffold for the sham control at 4 days, and 3582.3±325.3 ng/scaffold for the ultrasound-treated group and 2825.7±134.3 ng/scaffold for the sham control at 7 days. There was a significant difference in the dsDNA content between the ultrasound- and sham-treated groups at 4 and 7 days. The ultrasound-treated group with the SiC construct showed a 9% (p=0.00029) and 27% (p=0.00017) increase in the average dsDNA content at 4 and 7 days over the sham control group, respectively. Alkaline phosphatase activity was significantly increased by the treatment of ultrasound at 4 (p=0.012) and 7 days (p=0.035). These results suggested that ultrasound treatment with low-intensity acoustic energy facilitated the cellular ingrowth and enhanced the proliferation and early differentiation of osteoblasts in SiC scaffolds. PMID:24935158

C 3-symmetric opioid scaffolds are pH-responsive DNA condensation agents.

PubMed

McStay, Natasha; Molphy, Zara; Coughlan, Alan; Cafolla, Attilio; McKee, Vickie; Gathergood, Nicholas; Kellett, Andrew

2017-01-25

Herein we report the synthesis of tripodal C 3 -symmetric opioid scaffolds as high-affinity condensation agents of duplex DNA. Condensation was achieved on both supercoiled and canonical B-DNA structures and identified by agarose electrophoresis, viscosity, turbidity and atomic force microscopy (AFM) measurements. Structurally, the requirement of a tris-opioid scaffold for condensation is demonstrated as both di- (C 2 -symmetric) and mono-substituted (C 1 -symmetric) mesitylene-linked opioid derivatives poorly coordinate dsDNA. Condensation, observed by toroidal and globule AFM aggregation, arises from surface-binding ionic interactions between protonated, cationic, tertiary amine groups on the opioid skeleton and the phosphate nucleic acid backbone. Indeed, by converting the 6-hydroxyl group of C 3 -morphine ( MC3: ) to methoxy substituents in C 3 -heterocodeine ( HC3: ) and C 3 -oripavine ( OC3: ) molecules, dsDNA compaction is retained thus negating the possibility of phosphate-hydroxyl surface-binding. Tripodal opioid condensation was identified as pH dependent and strongly influenced by ionic strength with further evidence of cationic amine-phosphate backbone coordination arising from thermal melting analysis and circular dichroism spectroscopy, with compaction also witnessed on synthetic dsDNA co-polymers poly[d(A-T) 2 ] and poly[d(G-C) 2 ]. On-chip microfluidic analysis of DNA condensed by C 3 -agents provided concentration-dependent protection (inhibition) to site-selective excision by type II restriction enzymes: BamHI, HindIII, SalI and EcoRI, but not to the endonuclease DNase I. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold.

PubMed

Lin, Hsin-Yi; Chen, Hsin-Hung; Chang, Shih-Hsin; Ni, Tsung-Sheng

2013-01-01

Scaffolds made of chitosan nanofibers are often too mechanically weak for their application and often their manufacturing processes involve the use of harmful and flammable organic solvents. In the attempt to improve the mechanical properties of nanofibrous scaffolds made of chitosan without the use of harmful chemicals, pectin, an anionic polymer was blended with chitosan, a cationic polymer, to form a polyelectrolyte complex and electrospun into nanofibers for the first time. The electrospun chitosan-pectin scaffolds, when compared to electrospun chitosan scaffolds, had a 58% larger diameter, a 21% higher Young's modulus, a 162% larger strain at break, and a 104% higher ultimate tensile strength. Compared to the chitosan scaffolds, the chitosan-pectin scaffolds' swelling ratios decreased by 55% after 60 min in a saline solution and more quickly released the preloaded tetracycline HCl. The L929 fibroblast cells proliferated slightly slower on the chitosan-pectin scaffolds than on the chitosan scaffolds. Nonetheless, cells on both materials deposited similar levels of extracellular type I collagen on a per DNA basis. In conclusion, a novel chitosan-pectin nanofibrous scaffold with superior mechanical properties than a chitosan nanofibrous scaffold was successfully made without the use of harmful solvents.

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

PubMed

MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

2015-09-14

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Preparation and Characterization of a Novel Decellularized Fibrocartilage "Book" Scaffold for Use in Tissue Engineering.

PubMed

Guo, Liyun; Qu, Jin; Zheng, Cheng; Cao, Yong; Zhang, Tao; Lu, Hongbin; Hu, Jianzhong

2015-01-01

At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm) with 10 layers, each layer (akin to a page of a book) with a thickness of 100-μm. These fibrocartilage "book" scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel "book" fibrocartilage scaffold could have potential applications in tissue engineering.

Preparation and Characterization of a Novel Decellularized Fibrocartilage “Book” Scaffold for Use in Tissue Engineering

PubMed Central

Guo, Liyun; Qu, Jin; Zheng, Cheng; Cao, Yong; Zhang, Tao; Lu, Hongbin; Hu, Jianzhong

2015-01-01

At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm) with 10 layers, each layer (akin to a page of a book) with a thickness of 100-μm. These fibrocartilage “book” scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel “book” fibrocartilage scaffold could have potential applications in tissue engineering. PMID:26636672

Hierarchical Scaffolding With Bambus

PubMed Central

Pop, Mihai; Kosack, Daniel S.; Salzberg, Steven L.

2004-01-01

The output of a genome assembler generally comprises a collection of contiguous DNA sequences (contigs) whose relative placement along the genome is not defined. A procedure called scaffolding is commonly used to order and orient these contigs using paired read information. This ordering of contigs is an essential step when finishing and analyzing the data from a whole-genome shotgun project. Most recent assemblers include a scaffolding module; however, users have little control over the scaffolding algorithm or the information produced. We thus developed a general-purpose scaffolder, called Bambus, which affords users significant flexibility in controlling the scaffolding parameters. Bambus was used recently to scaffold the low-coverage draft dog genome data. Most significantly, Bambus enables the use of linking data other than that inferred from mate-pair information. For example, the sequence of a completed genome can be used to guide the scaffolding of a related organism. We present several applications of Bambus: support for finishing, comparative genomics, analysis of the haplotype structure of genomes, and scaffolding of a mammalian genome at low coverage. Bambus is available as an open-source package from our Web site. PMID:14707177

Hierarchical scaffolding with Bambus.

PubMed

Pop, Mihai; Kosack, Daniel S; Salzberg, Steven L

2004-01-01

The output of a genome assembler generally comprises a collection of contiguous DNA sequences (contigs) whose relative placement along the genome is not defined. A procedure called scaffolding is commonly used to order and orient these contigs using paired read information. This ordering of contigs is an essential step when finishing and analyzing the data from a whole-genome shotgun project. Most recent assemblers include a scaffolding module; however, users have little control over the scaffolding algorithm or the information produced. We thus developed a general-purpose scaffolder, called Bambus, which affords users significant flexibility in controlling the scaffolding parameters. Bambus was used recently to scaffold the low-coverage draft dog genome data. Most significantly, Bambus enables the use of linking data other than that inferred from mate-pair information. For example, the sequence of a completed genome can be used to guide the scaffolding of a related organism. We present several applications of Bambus: support for finishing, comparative genomics, analysis of the haplotype structure of genomes, and scaffolding of a mammalian genome at low coverage. Bambus is available as an open-source package from our Web site.

Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves.

PubMed

Theodoridis, Karolina; Müller, Janina; Ramm, Robert; Findeisen, Katja; Andrée, Birgit; Korossis, Sotirios; Haverich, Axel; Hilfiker, Andres

2016-10-01

Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

NASA Technical Reports Server (NTRS)

Ishaug-Riley, S. L.; Crane-Kruger, G. M.; Yaszemski, M. J.; Mikos, A. G.

1998-01-01

Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study demonstrates that PLGA foams are suitable substrates for osteoblast growth and differentiated function independent of cell source.

Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics.

PubMed

Paula, Débora P; Linard, Benjamin; Andow, David A; Sujii, Edison R; Pires, Carmen S S; Vogler, Alfried P

2015-07-01

DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR-based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross-reactions with the predator and related species genomes. Here, we use PCR-free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding, multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h(-1) respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h(-1) , respectively). However, the facultative aphid symbionts Hamiltonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1-2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts. © 2014 John Wiley & Sons Ltd.

Characterization of decellularized scaffold derived from porcine meniscus for tissue engineering applications

NASA Astrophysics Data System (ADS)

Gao, Shuang; Yuan, Zhiguo; Xi, Tingfei; Wei, Xiaojuan; Guo, Quanyi

2016-06-01

Menisci are fundamental fibrocartilaginous organs in knee joints. The injury in meniscus can impair normal knee function and predisposes patients to osteoarthritis. This study prepared decellularized meniscus scaffolds using a 1% (w/w) sodium dodecyl sulfate solution and sufficient rinsing steps. Complete cell removal was verified by hematoxylin and eosin staining and DNA content assay. Decellularized menisci had accordant tension properties to intact ones, but with declined compression properties. This occurred because the collagen fiber was not damaged but glycosaminoglycans was significantly lost during the decellularization process, which was confirmed by biochemical assay and histology staining. In vitro cytotoxicity assay demonstrated that decellularized meniscus scaffolds have no toxicity on L929 murine fibroblasts and porcine chondrocytes. Further experiment showed that porcine chondrocytes could adhere and proliferate on the scaffold surface, and some cells even could infiltrate into the scaffold. All results showed the potential of this decellularized meniscus to be the scaffolds in tissue engineering.

Preparation of porous collagen/hyaluronic acid hybrid scaffolds for biomimetic functionalization through biochemical binding affinity.

PubMed

Lee, Su Jin; Kim, So Yeon; Lee, Young Moo

2007-08-01

This study demonstrated the feasibility of introducing an avidin-biotin system to three-dimensional and highly porous scaffolds for the purpose of designing scaffolds that have binding affinity with bioactive molecules for various biomimetic modifications. Porous hybrid scaffolds composed of collagen and hyaluronic acid (HA) were prepared by a novel overrun process. The overrun-processed scaffolds showed a uniform dual-pore structure because of the injection of gas bubbles and ice recrystallization during the fabrication process and had a higher porosity than scaffolds prepared by a conventional freeze-drying method. The mechanical strength and biodegradation kinetics were controlled by the method of preparation and the composition of collagen/HA. Collagen/HA scaffolds did not show any significant adverse effects on cell viability even after 10 days of incubation. The fibroblasts cultured in the overrun-processed scaffolds were widely distributed and had proliferated on the surfaces of the macropores in the scaffolds, whereas the cells that were seeded in the freeze-dried scaffolds had attached mainly on the dense surface of the scaffolds. As the collagen content in the scaffolds increased, the cellular ingrowth into the inner pores of the scaffolds decreased because of the high affinity between the collagen and the cells. Measurements obtained via confocal microscopy revealed that the porous collagen/HA scaffolds could be functionalized with the biotin by incorporating avidin. Therefore, the present biotinylation approach may allow the incorporation of various bioactive molecules (DNA, growth factors, drug, peptide, etc) into the three-dimensional porous scaffolds.

Tissue Engineering of Ureteral Grafts: Preparation of Biocompatible Crosslinked Ureteral Scaffolds of Porcine Origin

PubMed Central

Koch, Holger; Hammer, Niels; Ossmann, Susann; Schierle, Katrin; Sack, Ulrich; Hofmann, Jörg; Wecks, Mike; Boldt, Andreas

2015-01-01

The surgical reconstruction of ureteric defects is often associated with post-operative complications and requires additional medical care. Decellularized ureters originating from porcine donors could represent an alternative therapy. Our aim was to investigate the possibility of manufacturing decellularized ureters, the characteristics of the extracellular matrix (ECM) and the biocompatibility of these grafts in vitro/in vivo after treatment with different crosslinking agents. To achieve these goals, native ureters were obtained from pigs and were decellularized. The success of decellularization and the ECM composition were characterized by (immuno)histological staining methods and a DNA-assay. In vitro: scaffolds were crosslinked either with carbodiimide (CDI), genipin (GP), glutaraldehyde, left chemically untreated or were lyophilized. Scaffolds in each group were reseeded with Caco2, LS48, 3T3 cells, or native rat smooth muscle cells (SMC). After 2 weeks, the number of ingrown cells was quantified. In vivo: crosslinked scaffolds were implanted subcutaneously into rats and the type of infiltrating cells were determined after 1, 9, and 30 days. After decellularization, scaffold morphology and composition of ECM were maintained, all cellular components were removed, DNA destroyed and strongly reduced. In vitro: GP and CDI scaffolds revealed a higher number of ingrown 3T3 and SMC cells as compared to untreated scaffolds. In vivo: at day 30, implants were predominantly infiltrated by fibroblasts and M2 anti-inflammatory macrophages. A maximum of MMP3 was observed in the CDI group at day 30. TIMP1 was below the detection limit. In this study, we demonstrated the potential of decellularization to create biocompatible porcine ureteric grafts, whereas a CDI-crosslink may facilitate the remodeling process. The use of decellularized ureteric grafts may represent a novel therapeutic method in reconstruction of ureteric defects. PMID:26157796

Decellularized Rat Lung Scaffolds Using Sodium Lauryl Ether Sulfate for Tissue Engineering.

PubMed

Ma, Jinhui; Ju, Zhihai; Yu, Jie; Qiao, Yeru; Hou, Chenwei; Wang, Chen; Hei, Feilong

Perfusion decellularization with detergents is effective to maintain the architecture and proteins of extracellular matrix (ECM) for use in the field of lung tissue engineering (LTE). However, it is unclear which detergent is ideal to produce an acellular lung scaffold. In this study, we obtained two decellularized rat lung scaffolds using a novel detergent sodium lauryl ether sulfate (SLES) and a conventional detergent sodium dodecyl sulfate (SDS). Both decellularized lung scaffolds were assessed by histology, immunohistochemistry, scanning electron microscopy, DNA quantification, sulfated glycosaminoglycans (GAGs) quantification and western blot. Subsequently, the scaffolds were implanted subcutaneously in rats for 6 weeks and were evaluated via hematoxylin and eosin staining and Masson staining. Results indicated that SLES was effective to remove cells; moreover, lungs decellularized with SLES showed better preservation of sulfated GAGs, lung architecture, and ECM proteins than SDS. After 6 weeks, SLES scaffolds demonstrated a significantly greater potential for cell infiltration and blood vessel formation compared with SDS scaffolds. Taken together, we conclude that SLES is a promising detergent to produce an acellular scaffold using LTE for eventual transplantation.

ScaffoldSeq: Software for characterization of directed evolution populations.

PubMed

Woldring, Daniel R; Holec, Patrick V; Hackel, Benjamin J

2016-07-01

ScaffoldSeq is software designed for the numerous applications-including directed evolution analysis-in which a user generates a population of DNA sequences encoding for partially diverse proteins with related functions and would like to characterize the single site and pairwise amino acid frequencies across the population. A common scenario for enzyme maturation, antibody screening, and alternative scaffold engineering involves naïve and evolved populations that contain diversified regions, varying in both sequence and length, within a conserved framework. Analyzing the diversified regions of such populations is facilitated by high-throughput sequencing platforms; however, length variability within these regions (e.g., antibody CDRs) encumbers the alignment process. To overcome this challenge, the ScaffoldSeq algorithm takes advantage of conserved framework sequences to quickly identify diverse regions. Beyond this, unintended biases in sequence frequency are generated throughout the experimental workflow required to evolve and isolate clones of interest prior to DNA sequencing. ScaffoldSeq software uniquely handles this issue by providing tools to quantify and remove background sequences, cluster similar protein families, and dampen the impact of dominant clones. The software produces graphical and tabular summaries for each region of interest, allowing users to evaluate diversity in a site-specific manner as well as identify epistatic pairwise interactions. The code and detailed information are freely available at http://research.cems.umn.edu/hackel. Proteins 2016; 84:869-874. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

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

Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinitymore » for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.« less

Self-assembled nanoscale DNA-porphyrin complex for artificial light harvesting.

PubMed

Woller, Jakob G; Hannestad, Jonas K; Albinsson, Bo

2013-02-20

Mimicking green plants' and bacteria's extraordinary ability to absorb a vast number of photons and harness their energy is a longstanding goal in artificial photosynthesis. Resonance energy transfer among donor dyes has been shown to play a crucial role on the overall transfer of energy in the natural systems. Here, we present artificial, self-assembled, light-harvesting complexes consisting of DNA scaffolds, intercalated YO-PRO-1 (YO) donor dyes and a porphyrin acceptor anchored to a lipid bilayer, conceptually mimicking the natural light-harvesting systems. A model system consisting of 39-mer duplex DNA in a linear wire configuration with the porphyrin attached in the middle of the wire is primarily investigated. Utilizing intercalated donor fluorophores to sensitize the excitation of the porphyrin acceptor, we obtain an effective absorption coefficient 12 times larger than for direct excitation of the porphyrin. On the basis of steady-state and time-resolved emission measurements and Markov chain simulations, we show that YO-to-YO resonance energy transfer substantially contributes to the overall flow of energy to the porphyrin. This increase is explained through energy migration along the wire allowing the excited state energy to transfer to positions closer to the porphyrin. The versatility of DNA as a structural material is demonstrated through the construction of a more complex, hexagonal, light-harvesting scaffold yielding further increase in the effective absorption coefficient. Our results show that, by using DNA as a scaffold, we are able to arrange chromophores on a nanometer scale and in this way facilitate the assembly of efficient light-harvesting systems.

Parallel fabrication of macroporous scaffolds.

PubMed

Dobos, Andrew; Grandhi, Taraka Sai Pavan; Godeshala, Sudhakar; Meldrum, Deirdre R; Rege, Kaushal

2018-07-01

Scaffolds generated from naturally occurring and synthetic polymers have been investigated in several applications because of their biocompatibility and tunable chemo-mechanical properties. Existing methods for generation of 3D polymeric scaffolds typically cannot be parallelized, suffer from low throughputs, and do not allow for quick and easy removal of the fragile structures that are formed. Current molds used in hydrogel and scaffold fabrication using solvent casting and porogen leaching are often single-use and do not facilitate 3D scaffold formation in parallel. Here, we describe a simple device and related approaches for the parallel fabrication of macroporous scaffolds. This approach was employed for the generation of macroporous and non-macroporous materials in parallel, in higher throughput and allowed for easy retrieval of these 3D scaffolds once formed. In addition, macroporous scaffolds with interconnected as well as non-interconnected pores were generated, and the versatility of this approach was employed for the generation of 3D scaffolds from diverse materials including an aminoglycoside-derived cationic hydrogel ("Amikagel"), poly(lactic-co-glycolic acid) or PLGA, and collagen. Macroporous scaffolds generated using the device were investigated for plasmid DNA binding and cell loading, indicating the use of this approach for developing materials for different applications in biotechnology. Our results demonstrate that the device-based approach is a simple technology for generating scaffolds in parallel, which can enhance the toolbox of current fabrication techniques. © 2018 Wiley Periodicals, Inc.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

PubMed

Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

2013-01-18

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima

PubMed Central

Green, Jack E.; Dalíková, Martina; Sahara, Ken; Marec, František; Akam, Michael

2016-01-01

We show that the geophilomorph centipede Strigamia maritima possesses an XX/XY system of sex chromosomes, with males being the heterogametic sex. This is, to our knowledge, the first report of sex chromosomes in any geophilomorph centipede. Using the recently assembled Strigamia genome sequence, we identified a set of scaffolds differentially represented in male and female DNA sequence. Using quantitative real-time PCR, we confirmed that three candidate X chromosome-derived scaffolds are present at approximately twice the copy number in females as in males. Furthermore, we confirmed that six candidate Y chromosome-derived scaffolds contain male-specific sequences. Finally, using this molecular information, we designed an X chromosome-specific DNA probe and performed fluorescent in situ hybridization against mitotic and meiotic chromosome spreads to identify the Strigamia XY sex-chromosome pair cytologically. We found that the X and Y chromosomes are recognizably different in size during the early pachytene stage of meiosis, and exhibit incomplete and delayed pairing. PMID:26919730

Structural and biomechanical characterizations of porcine myocardial extracellular matrix

PubMed Central

Wang, Bo; Tedder, Mary E.; Perez, Clara E.; Wang, Guangjun; de Jongh Curry, Amy L.; To, Filip; Elder, Steven H.; Williams, Lakiesha N.; Simionescu, Dan T.; Liao, Jun

2012-01-01

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson’s trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat’s pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering. PMID:22584822

Fabrication and characterization of a rapid prototyped tissue engineering scaffold with embedded multicomponent matrix for controlled drug release.

PubMed

Chen, Muwan; Le, Dang Q S; Hein, San; Li, Pengcheng; Nygaard, Jens V; Kassem, Moustapha; Kjems, Jørgen; Besenbacher, Flemming; Bünger, Cody

2012-01-01

Bone tissue engineering implants with sustained local drug delivery provide an opportunity for better postoperative care for bone tumor patients because these implants offer sustained drug release at the tumor site and reduce systemic side effects. A rapid prototyped macroporous polycaprolactone scaffold was embedded with a porous matrix composed of chitosan, nanoclay, and β-tricalcium phosphate by freeze-drying. This composite scaffold was evaluated on its ability to deliver an anthracycline antibiotic and to promote formation of mineralized matrix in vitro. Scanning electronic microscopy, confocal imaging, and DNA quantification confirmed that immortalized human bone marrow-derived mesenchymal stem cells (hMSC-TERT) cultured in the scaffold showed high cell viability and growth, and good cell infiltration to the pores of the scaffold. Alkaline phosphatase activity and osteocalcin staining showed that the scaffold was osteoinductive. The drug-release kinetics was investigated by loading doxorubicin into the scaffold. The scaffolds comprising nanoclay released up to 45% of the drug for up to 2 months, while the scaffold without nanoclay released 95% of the drug within 4 days. Therefore, this scaffold can fulfill the requirements for both bone tissue engineering and local sustained release of an anticancer drug in vitro. These results suggest that the scaffold can be used clinically in reconstructive surgery after bone tumor resection. Moreover, by changing the composition and amount of individual components, the scaffold can find application in other tissue engineering areas that need local sustained release of drug.

Fabrication and characterization of a rapid prototyped tissue engineering scaffold with embedded multicomponent matrix for controlled drug release

PubMed Central

Chen, Muwan; Le, Dang QS; Hein, San; Li, Pengcheng; Nygaard, Jens V; Kassem, Moustapha; Kjems, Jørgen; Besenbacher, Flemming; Bünger, Cody

2012-01-01

Bone tissue engineering implants with sustained local drug delivery provide an opportunity for better postoperative care for bone tumor patients because these implants offer sustained drug release at the tumor site and reduce systemic side effects. A rapid prototyped macroporous polycaprolactone scaffold was embedded with a porous matrix composed of chitosan, nanoclay, and β-tricalcium phosphate by freeze-drying. This composite scaffold was evaluated on its ability to deliver an anthracycline antibiotic and to promote formation of mineralized matrix in vitro. Scanning electronic microscopy, confocal imaging, and DNA quantification confirmed that immortalized human bone marrow-derived mesenchymal stem cells (hMSC-TERT) cultured in the scaffold showed high cell viability and growth, and good cell infiltration to the pores of the scaffold. Alkaline phosphatase activity and osteocalcin staining showed that the scaffold was osteoinductive. The drug-release kinetics was investigated by loading doxorubicin into the scaffold. The scaffolds comprising nanoclay released up to 45% of the drug for up to 2 months, while the scaffold without nanoclay released 95% of the drug within 4 days. Therefore, this scaffold can fulfill the requirements for both bone tissue engineering and local sustained release of an anticancer drug in vitro. These results suggest that the scaffold can be used clinically in reconstructive surgery after bone tumor resection. Moreover, by changing the composition and amount of individual components, the scaffold can find application in other tissue engineering areas that need local sustained release of drug. PMID:22904634

Defined presentation of carbohydrates on a duplex DNA scaffold.

PubMed

Schlegel, Mark K; Hütter, Julia; Eriksson, Magdalena; Lepenies, Bernd; Seeberger, Peter H

2011-12-16

A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enamel matrix derivative enhances tissue formation around scaffolds used for tissue engineering of ligaments.

PubMed

Messenger, Michael P; Raïf, El M; Seedhom, Bahaa B; Brookes, Steven J

2010-02-01

The following in vitro translational study investigated whether enamel matrix derivative (EMD), an approved biomimetic treatment for periodontal disease (Emdogain) and hard-to-heal wounds (Xelma), enhanced synovial cell colonization and protein synthesis around a scaffold used clinically for in situ tissue engineering of the torn anterior cruciate ligament (ACL). Synovial cells were enzymatically extracted from bovine synovium and dynamically seeded onto polyethylene terephthalate (PET) scaffolds. The cells were cultured in low-serum medium (0.5% FBS) for 4 weeks with either a single administration of EMD at the start of the 4 week period or multiple administrations of EMD at regular intervals throughout the 4 weeks. Samples were harvested and evaluated using the Hoechst DNA assay, BCA protein assay, cresolphthalein complexone calcium assay, SDS-PAGE, ELISA and electron microscopy. A significant increase in cell number (DNA) (p < 0.01), protein content (p < 0.01) and TGFbeta1 synthesis (p < 0.01) was observed with multiple administrations of EMD. Additionally, SDS-PAGE showed an increase in high molecular weight proteins, characteristic of the fibril-forming collagens. Electron microscopy supported these findings, showing that scaffolds treated with multiple administrations of EMD were heavily coated with cells and extracellular matrix (ECM) that enveloped the fibres. Multiple administrations of EMD to synovial cell-seeded scaffolds enhanced the formation of tissue in vitro. Additionally, it was shown that EMD enhanced TGFbeta1 synthesis of synovial cells, suggesting a potential mode of action for EMD's capacity to stimulate tissue regeneration.

Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2017-05-24

A novel DNA tetrahedron-structured electrochemiluminescence (ECL) platform for bioanalysis with programmable DNA cyclic amplification was developed. In this work, glucose oxidase (GOD) was labeled to a DNA sequence (S) as functional conjugation (GOD-S), which could hybridize with other DNA sequences (L and P) to form GOD-S:L:P probe. In the presence of target DNA and a help DNA (A), the programmable DNA cyclic amplification was activated and released GOD-S via toehold-mediated strand displacement. Then, the obtained GOD-S was further immobilized on the DNA tetrahedral scaffolds with a pendant capture DNA and Ru(bpy) 3 2+ -conjugated silica nanoparticles (RuSi NPs) decorated on the electrode surface. Thus, the amount of GOD-S assembled on the electrode surface depended on the concentration of target DNA and GOD could catalyze glucose to generate H 2 O 2 in situ. The ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by the presence of H 2 O 2 . By integrating the programmable DNA cyclic amplification and in situ generating H 2 O 2 as Ru(bpy) 3 2+ ECL quencher, a sensitive DNA tetrahedron-structured ECL sensing platform was proposed for DNA detection. Under optimized conditions, this biosensor showed a wide linear range from 100 aM to 10 pM with a detection limit of 40 aM, indicating a promising application in DNA analysis. Furthermore, by labeling GOD to different recognition elements, the proposed strategy could be used for the detection of various targets. Thus, this programmable cascade amplification strategy not only retains the high selectivity and good capturing efficiency of tetrahedral-decorated electrode surface but also provides potential applications in the construction of ECL biosensor.

Osteoinductive-nanoscaled silk/HA composite scaffolds for bone tissue engineering application.

PubMed

Huang, Xiaowei; Bai, Shumeng; Lu, Qiang; Liu, Xi; Liu, Shanshan; Zhu, Hesun

2015-10-01

Osteoinductive silk/hydroxyapatite (HA) composite scaffolds for bone regeneration were prepared by combining silk with HA/silk core-shell nanoparticles. The HA/silk nanoparticles were directly dispersed in silk solution to form uniform silk/HA blend and then composite scaffolds after a freeze-drying process. The HA/silk nanoparticles uniformly distributed in silk scaffolds at nanometer scale at varying HA content up to 40%, and substantially improved the compressive strength of the scaffolds produced. Rat bone mesenchymal stem cells (rBMSCs) were cultured in these scaffolds and cell proliferation was analyzed by confocal microscopy and DNA assay. Gene expression and biochemical assays were employed to study the influence of increasing HA/silk nanoparticles on in vitro osteogenic differentiation of rBMSCs. Increasing HA/silk nanoparticles inside silk scaffolds improved the growth and osteogenic capability of rBMSCs in the absence of osteogenic growth factors, and also significantly increased the calcium and collagen I deposition. In addition, compared to silk/HA composite scaffolds containing HA aggregates, the scaffolds loaded with HA/silk nanoparticles showed remarkably higher stiffness and better osteogenic property at same HA content, implying a preferable microenvironment for rBMSCs. These results suggest that the osteogenic property as well as mechanical property of silk/HA scaffolds could be further improved through fabricating their structure and topography at nanometer scale, providing more suitable systems for bone regeneration. © 2014 Wiley Periodicals, Inc.

The Human Ligase IIIα-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA*

PubMed Central

Rashid, Ishtiaque; Tomkinson, Alan E.; Pederson, David S.

2017-01-01

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin. PMID:28184006

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds.

PubMed

Butler, Colin R; Hynds, Robert E; Crowley, Claire; Gowers, Kate H C; Partington, Leanne; Hamilton, Nicholas J; Carvalho, Carla; Platé, Manuela; Samuel, Edward R; Burns, Alan J; Urbani, Luca; Birchall, Martin A; Lowdell, Mark W; De Coppi, Paolo; Janes, Sam M

2017-04-01

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Composite poly(l-lactic-acid)/silk fibroin scaffold prepared by electrospinning promotes chondrogenesis for cartilage tissue engineering.

PubMed

Li, Zhengqiang; Liu, Peng; Yang, Ting; Sun, Ying; You, Qi; Li, Jiale; Wang, Zilin; Han, Bing

2016-05-01

Nanofibrous materials produced by electrospinning have attracted considerable attention from researchers in regenerative medicine. A combination of nanofibrous scaffold and chondrocytes is considered promising for repair of cartilage defect or damage. In the present study, we fabricated a poly(l-lactic-acid) (PLLA)/silk fibroin (SF) nanofibrous scaffold by electrospinning and evaluated its chondrogenic potential. The PLLA/SF nanofibers were characterized for diameter, surface wettability, swelling ratio, and tensile strength. Throughin vitroexperiments, PLLA/SF scaffold-chondrocyte interactions were investigated relative to the unmodified PLLA scaffold with regard to cellular adhesion, spreading, and proliferation by scanning electron microscopy and confocal laser scanning microscopy, and through analyses of DNA, sulfated glycosaminoglycan, and collagen. In addition, hematoxylin-eosin and Alcian blue-nuclear fast red staining were used to observe growth of chondrocytes, and secretion and distribution of cartilage-specific extracellular matrices in the scaffolds. Expressions of cartilage-related genes (collagen II, aggrecan, sox9, collagen I, and collagen X) were detected by real-time quantitative PCR. The PLLA/SF scaffold had better hydrophilicity, and could support chondrocytes adhesion and spreading more effectively than the unmodified PLLA scaffold. Chondrocytes secreted more cartilage-specific extracellular matrices and maintained their phenotype on the PLLA/SF scaffold. So it is concluded that the PLLA/SF scaffold is more conducive toin vitroformation of cartilage-like new tissues than the unmodified PLLA scaffold, and may be a promising material in cartilage tissue engineering. © The Author(s) 2016.

Periodontal tissue engineering by nano beta-tricalcium phosphate scaffold and fibroblast growth factor-2 in one-wall infrabony defects of dogs.

PubMed

Ogawa, K; Miyaji, H; Kato, A; Kosen, Y; Momose, T; Yoshida, T; Nishida, E; Miyata, S; Murakami, S; Takita, H; Fugetsu, B; Sugaya, T; Kawanami, M

2016-12-01

Nanoparticle bioceramics are being investigated for biomedical applications. We fabricated a regenerative scaffold comprising type I collagen and beta-tricalcium phosphate (β-TCP) nanoparticles. Fibroblast growth factor-2 (FGF-2) is a bioeffective signaling molecule that stimulates cell proliferation and wound healing. This study examined the effects, on bioactivity, of a nano-β-TCP/collagen scaffold loaded with FGF-2, particularly on periodontal tissue wound healing. Beta-tricalcium phosphate was pulverized into nanosize particles (84 nm) and was then dispersed. A nano-β-TCP scaffold was prepared by coating the surface of a collagen scaffold with a nanosize β-TCP dispersion. Scaffolds were characterized using scanning electron microscopy, compressive testing, cell seeding and rat subcutaneous implant testing. Then, nano-β-TCP scaffold, nano-β-TCP scaffold loaded with FGF-2 and noncoated collagen scaffold were implanted into a dog one-wall infrabony defect model. Histological observations were made at 10 d and 4 wk postsurgery. Scanning electron microscopy images show that TCP nanoparticles were attached to collagen fibers. The nano-β-TCP scaffold showed higher compressive strength and cytocompatibility compared with the noncoated collagen scaffold. Rat subcutaneous implant tests showed that the DNA contents of infiltrating cells in the nano-β-TCP scaffold and the FGF-2-loaded scaffold were approximately 2.8-fold and 3.7-fold greater, respectively, than in the collagen scaffold. Histological samples from the periodontal defect model showed about five-fold greater periodontal tissue repair following implantation of the nano-β-TCP scaffold loaded with FGF-2 compared with the collagen scaffold. The β-TCP nanoparticle coating strongly improved the collagen scaffold bioactivity. Nano-β-TCP scaffolds containing FGF-2 are anticipated for use in periodontal tissue engineering. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Graphene oxide scaffold accelerates cellular proliferative response and alveolar bone healing of tooth extraction socket.

PubMed

Nishida, Erika; Miyaji, Hirofumi; Kato, Akihito; Takita, Hiroko; Iwanaga, Toshihiko; Momose, Takehito; Ogawa, Kosuke; Murakami, Shusuke; Sugaya, Tsutomu; Kawanami, Masamitsu

2016-01-01

Graphene oxide (GO) consisting of a carbon monolayer has been widely investigated for tissue engineering platforms because of its unique properties. For this study, we fabricated a GO-applied scaffold and assessed the cellular and tissue behaviors in the scaffold. A preclinical test was conducted to ascertain whether the GO scaffold promoted bone induction in dog tooth extraction sockets. For this study, GO scaffolds were prepared by coating the surface of a collagen sponge scaffold with 0.1 and 1 µg/mL GO dispersion. Scaffolds were characterized using scanning electron microscopy (SEM), physical testing, cell seeding, and rat subcutaneous implant testing. Then a GO scaffold was implanted into a dog tooth extraction socket. Histological observations were made at 2 weeks postsurgery. SEM observations show that GO attached to the surface of collagen scaffold struts. The GO scaffold exhibited an interconnected structure resembling that of control subjects. GO application improved the physical strength, enzyme resistance, and adsorption of calcium and proteins. Cytocompatibility tests showed that GO application significantly increased osteoblastic MC3T3-E1 cell proliferation. In addition, an assessment of rat subcutaneous tissue response revealed that implantation of 1 µg/mL GO scaffold stimulated cellular ingrowth behavior, suggesting that the GO scaffold exhibited good biocompatibility. The tissue ingrowth area and DNA contents of 1 µg/mL GO scaffold were, respectively, approximately 2.5-fold and 1.4-fold greater than those of the control. Particularly, the infiltration of ED2-positive (M2) macrophages and blood vessels were prominent in the GO scaffold. Dog bone-formation tests showed that 1 µg/mL GO scaffold implantation enhanced bone formation. New bone formation following GO scaffold implantation was enhanced fivefold compared to that in control subjects. These results suggest that GO was biocompatible and had high bone-formation capability for the scaffold. The GO scaffold is expected to be beneficial for bone tissue engineering therapy.

Biotechnological mass production of DNA origami

NASA Astrophysics Data System (ADS)

Praetorius, Florian; Kick, Benjamin; Behler, Karl L.; Honemann, Maximilian N.; Weuster-Botz, Dirk; Dietz, Hendrik

2017-12-01

DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising ‘cassettes’, with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.

Biotechnological mass production of DNA origami.

PubMed

Praetorius, Florian; Kick, Benjamin; Behler, Karl L; Honemann, Maximilian N; Weuster-Botz, Dirk; Dietz, Hendrik

2017-12-06

DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn 2+ -dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.

Design and construction of a DNA origami drug delivery system based on MPT64 antibody aptamer for tuberculosis treatment.

PubMed

Ranjbar, Reza; Hafezi-Moghadam, Mohammad Sadegh

2016-02-01

With all of the developments on infectious diseases, tuberculosis (TB) remains a cause of death among people. One of the most promising assembly techniques in nano-technology is "scaffolded DNA origami" to design and construct a nano-scale drug delivery system. Because of the global health problems of tuberculosis, the development of potent new anti-tuberculosis drug delivery system without cross-resistance with known anti-mycobacterial agents is urgently needed. The aim of this study was to design a nano-scale drug delivery system for TB treatment using the DNA origami method. In this study, we presented an experimental research on a DNA drug delivery system for treating Tuberculosis. TEM images were visualized with an FEI Tecnai T12 BioTWIN at 120 kV. The model was designed by caDNAno software and computational prediction of the 3D solution shape and its flexibility was calculated with a CanDo server. Synthesizing the product was imaged using transmission electron microscopy after negative-staining by uranyl formate. We constructed a multilayer 3D DNA nanostructure system by designing square lattice geometry with the scaffolded-DNA-origami method. With changes in the lock and key sequences, we recommend that this system be used for other infectious diseases to target the pathogenic bacteria.

Design and Synthesis of Biaryl DNA-Encoded Libraries.

PubMed

Ding, Yun; Franklin, G Joseph; DeLorey, Jennifer L; Centrella, Paolo A; Mataruse, Sibongile; Clark, Matthew A; Skinner, Steven R; Belyanskaya, Svetlana

2016-10-10

DNA-encoded library technology (ELT) is a powerful tool for the discovery of new small-molecule ligands to various protein targets. Here we report the design and synthesis of biaryl DNA-encoded libraries based on the scaffold of 5-formyl 3-iodobenzoic acid. Three reactions on DNA template, acylation, Suzuki-Miyaura coupling and reductive amination, were applied in the library synthesis. The three cycle library of 3.5 million diversity has delivered potent hits for phosphoinositide 3-kinase α (PI3Kα).

Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components

NASA Astrophysics Data System (ADS)

Ong, Luvena L.; Hanikel, Nikita; Yaghi, Omar K.; Grun, Casey; Strauss, Maximilian T.; Bron, Patrick; Lai-Kee-Him, Josephine; Schueder, Florian; Wang, Bei; Wang, Pengfei; Kishi, Jocelyn Y.; Myhrvold, Cameron; Zhu, Allen; Jungmann, Ralf; Bellot, Gaetan; Ke, Yonggang; Yin, Peng

2017-12-01

Nucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair ‘voxels’ that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.

Rational assembly of nanoparticle superlattices with designed lattice symmetries

DOEpatents

Gang, Oleg; Lu, Fang; Tagawa, Miho

2017-09-05

A method for lattice design via multivalent linkers (LDML) is disclosed that introduces a rationally designed symmetry of connections between particles in order to achieve control over the morphology of their assembly. The method affords the inclusion of different programmable interactions within one linker that allow an assembly of different types of particles. The designed symmetry of connections is preferably provided utilizing DNA encoding. The linkers may include fabricated "patchy" particles, DNA scaffold constructs and Y-shaped DNA linkers, anisotropic particles, which are preferably functionalized with DNA, multimeric protein-DNA complexes, and particles with finite numbers of DNA linkers.

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer.

PubMed

Garcia, Yolanda; Hemantkumar, Naik; Collighan, Russell; Griffin, Martin; Rodriguez-Cabello, Jose Carlos; Pandit, Abhay

2009-04-01

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time

PubMed Central

Williams, Gareth J.; Hammel, Michal; Radhakrishnan, Sarvan Kumar; Ramsden, Dale; Lees-Miller, Susan P.; Tainer, John A.

2014-01-01

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), 2) lesion processing, and 3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. PMID:24656613

Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

PubMed Central

2014-01-01

Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. PMID:24997498

A Rapid Method of Genomic Array Analysis of Scaffold/Matrix Attachment Regions (S/MARs) Identifies a 2.5-Mb Region of Enhanced Scaffold/Matrix Attachment at a Human Neocentromere

PubMed Central

Sumer, Huseyin; Craig, Jeffrey M.; Sibson, Mandy; Choo, K.H. Andy

2003-01-01

Human neocentromeres are fully functional centromeres that arise at previously noncentromeric regions of the genome. We have tested a rapid procedure of genomic array analysis of chromosome scaffold/matrix attachment regions (S/MARs), involving the isolation of S/MAR DNA and hybridization of this DNA to a genomic BAC/PAC array. Using this procedure, we have defined a 2.5-Mb domain of S/MAR-enriched chromatin that fully encompasses a previously mapped centromere protein-A (CENP-A)-associated domain at a human neocentromere. We have independently verified this procedure using a previously established fluorescence in situ hybridization method on salt-treated metaphase chromosomes. In silico sequence analysis of the S/MAR-enriched and surrounding regions has revealed no outstanding sequence-related predisposition. This study defines the S/MAR-enriched domain of a higher eukaryotic centromere and provides a method that has broad application for the mapping of S/MAR attachment sites over large genomic regions or throughout a genome. PMID:12840048

Mechanical properties and cell-culture characteristics of a polycaprolactone kagome-structure scaffold fabricated by a precision extruding deposition system.

PubMed

Lee, Se-Hwan; Cho, Yong Sang; Hong, Myoung Wha; Lee, Bu-Kyu; Park, Yongdoo; Park, Sang-Hyug; Kim, Young Yul; Cho, Young-Sam

2017-09-13

To enhance the mechanical properties of three-dimensional (3D) scaffolds used for bone regeneration in tissue engineering, many researchers have studied their structure and chemistry. In the structural engineering field, the kagome structure has been known to have an excellent relative strength. In this study, to enhance the mechanical properties of a synthetic polymer scaffold used for tissue engineering, we applied the 3D kagome structure to a porous scaffold for bone regeneration. Prior to fabricating the biocompatible-polymer scaffold, the ideal kagome structure, which was manufactured by a 3D printer of the digital light processing type, was compared with a grid-structure, which was used as the control group, using a compressive experiment. A polycaprolactone (PCL) kagome-structure scaffold was successfully fabricated by additive manufacturing using a 3D printer with a precision extruding deposition head. To assess the physical characteristics of the fabricated PCL-kagome-structure scaffold, we analyzed its porosity, pore size, morphological structure, surface roughness, compressive stiffness, and mechanical bending properties. The results showed that, the mechanical properties of proposed kagome-structure scaffold were superior to those of a grid-structure scaffold. Moreover, Sarcoma osteogenic (Saos-2) cells were used to evaluate the characteristics of in vitro cell proliferation. We carried out cell counting kit-8 (CCK-8) and DNA contents assays. Consequently, the cell proliferation of the kagome-structure scaffold was increased; this could be because the surface roughness of the kagome-structure scaffold enhances initial cell attachment.

Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold.

PubMed

Shlyahovsky, Bella; Li, Yang; Lioubashevski, Oleg; Elbaz, Johann; Willner, Itamar

2009-07-28

A series of logic gates, "AND", "OR", and "XOR", are designed using a DNA scaffold that includes four "footholds" on which the logic operations are activated. Two of the footholds represent input-recognition strands, and these are blocked by complementary nucleic acids, whereas the other two footholds are blocked by nucleic acids that include the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The logic gates are activated by either nucleic acid inputs that hybridize to the respective "footholds", or by low-molecular-weight inputs (adenosine monophosphate or cocaine) that yield the respective aptamer-substrate complexes. This results in the respective translocation of the blocking nucleic acids to the footholds carrying the HRP-mimicking DNAzyme sequence, and the concomitant release of the respective DNAzyme. The released product-strands then self-assemble into the hemin/G-quadruplex-HRP-mimicking DNAzyme that biocatalyzes the formation of a colored product and provides an output signal for the different logic gates. The principle of the logic operation is, then, implemented as a possible paradigm for future nanomedicine. The nucleic acid inputs that bind to the blocked footholds result in the translocation of the blocking nucleic acids to the respective footholds carrying the antithrombin aptamer. The released aptamer inhibits, then, the hydrolytic activity of thrombin. The system demonstrates the regulation of a biocatalytic reaction by a translator system activated on a DNA scaffold.

Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.

PubMed

Pati, Falguni; Song, Tae-Ha; Rijal, Girdhari; Jang, Jinah; Kim, Sung Won; Cho, Dong-Woo

2015-01-01

3D printing technique is the most sophisticated technique to produce scaffolds with tailorable physical properties. But, these scaffolds often suffer from limited biological functionality as they are typically made from synthetic materials. Cell-laid mineralized ECM was shown to be potential for improving the cellular responses and drive osteogenesis of stem cells. Here, we intend to improve the biological functionality of 3D-printed synthetic scaffolds by ornamenting them with cell-laid mineralized extracellular matrix (ECM) that mimics a bony microenvironment. We developed bone graft substitutes by using 3D printed scaffolds made from a composite of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and β-tricalcium phosphate (β-TCP) and mineralized ECM laid by human nasal inferior turbinate tissue-derived mesenchymal stromal cells (hTMSCs). A rotary flask bioreactor was used to culture hTMSCs on the scaffolds to foster formation of mineralized ECM. A freeze/thaw cycle in hypotonic buffer was used to efficiently decellularize (97% DNA reduction) the ECM-ornamented scaffolds while preserving its main organic and inorganic components. The ECM-ornamented 3D printed scaffolds supported osteoblastic differentiation of newly-seeded hTMSCs by upregulating four typical osteoblastic genes (4-fold higher RUNX2; 3-fold higher ALP; 4-fold higher osteocalcin; and 4-fold higher osteopontin) and increasing calcium deposition compared to bare 3D printed scaffolds. In vivo, in ectopic and orthotopic models in rats, ECM-ornamented scaffolds induced greater bone formation than that of bare scaffolds. These results suggest a valuable method to produce ECM-ornamented 3D printed scaffolds as off-the-shelf bone graft substitutes that combine tunable physical properties with physiological presentation of biological signals. Copyright © 2014 Elsevier Ltd. All rights reserved.

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins.

PubMed

Norman, Michael; Rivers, Caroline; Lee, Youn-Bok; Idris, Jalilah; Uney, James

2016-12-01

RNA-binding proteins play a central role in cellular metabolism by orchestrating the complex interactions of coding, structural and regulatory RNA species. The SAFB (scaffold attachment factor B) proteins (SAFB1, SAFB2 and SAFB-like transcriptional modulator, SLTM), which are highly conserved evolutionarily, were first identified on the basis of their ability to bind scaffold attachment region DNA elements, but attention has subsequently shifted to their RNA-binding and protein-protein interactions. Initial studies identified the involvement of these proteins in the cellular stress response and other aspects of gene regulation. More recently, the multifunctional capabilities of SAFB proteins have shown that they play crucial roles in DNA repair, processing of mRNA and regulatory RNA, as well as in interaction with chromatin-modifying complexes. With the advent of new techniques for identifying RNA-binding sites, enumeration of individual RNA targets has now begun. This review aims to summarise what is currently known about the functions of SAFB proteins. © 2016 The Author(s).

Programmed coherent coupling in a synthetic DNA-based excitonic circuit

NASA Astrophysics Data System (ADS)

Boulais, Étienne; Sawaya, Nicolas P. D.; Veneziano, Rémi; Andreoni, Alessio; Banal, James L.; Kondo, Toru; Mandal, Sarthak; Lin, Su; Schlau-Cohen, Gabriela S.; Woodbury, Neal W.; Yan, Hao; Aspuru-Guzik, Alán; Bathe, Mark

2018-02-01

Natural light-harvesting systems spatially organize densely packed chromophore aggregates using rigid protein scaffolds to achieve highly efficient, directed energy transfer. Here, we report a synthetic strategy using rigid DNA scaffolds to similarly program the spatial organization of densely packed, discrete clusters of cyanine dye aggregates with tunable absorption spectra and strongly coupled exciton dynamics present in natural light-harvesting systems. We first characterize the range of dye-aggregate sizes that can be templated spatially by A-tracts of B-form DNA while retaining coherent energy transfer. We then use structure-based modelling and quantum dynamics to guide the rational design of higher-order synthetic circuits consisting of multiple discrete dye aggregates within a DX-tile. These programmed circuits exhibit excitonic transport properties with prominent circular dichroism, superradiance, and fast delocalized exciton transfer, consistent with our quantum dynamics predictions. This bottom-up strategy offers a versatile approach to the rational design of strongly coupled excitonic circuits using spatially organized dye aggregates for use in coherent nanoscale energy transport, artificial light-harvesting, and nanophotonics.

Isothermal assembly of DNA origami structures using denaturing agents.

PubMed

Jungmann, Ralf; Liedl, Tim; Sobey, Thomas L; Shih, William; Simmel, Friedrich C

2008-08-06

DNA origami is one of the most promising recent developments in DNA self-assembly. It allows for the construction of arbitrary nanoscale patterns and objects by folding a long viral scaffold strand using a large number of short "staple" strands. Assembly is usually accomplished by thermal annealing of the DNA molecules in buffer solution. We here demonstrate that both 2D and 3D origami structures can be assembled isothermally by annealing the DNA strands in denaturing buffer, followed by a controlled reduction of denaturant concentration. This opens up origami assembly for the integration of temperature-sensitive components.

TOPBP1Dpb11 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1Rad9

PubMed Central

Sims, Jennie Rae; Freire, Raimundo

2017-01-01

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1–TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair. PMID:28228534

Luminescent Carbon Dot Mimics Assembled on DNA

PubMed Central

Chan, Ke Min; Xu, Wang; Kwon, Hyukin; Kietrys, Anna M.; Kool, Eric T.

2018-01-01

Nanometer-sized fragments of carbon in the form of multilayer graphene (“carbon dots”) have been under highly active study for applications in imaging. While offering advantages of low toxicity and photostability, such nanomaterials are inhomogeneous and have limited wavelengths of emission. Here we address these issues by assembling luminescent aromatic C16–C38 hydrocarbons together on a DNA scaffold in homogeneous, soluble molecular compounds. Monomer deoxyribosides of five different aromatic hydrocarbons were synthesized and assembled into a library of 1296 different tetramer compounds on PEG-polystyrene beads. These were screened for photostability and a range of emission colors using 365 nm excitation, observing visible light (>400 nm) emission. We identified a set of six oligomers (DNA-carbon assemblies, DNA-CAs) with exceptional photostability that emit from 400 to 680 nm in water, with Stokes shifts of up to 110 nm, quantum yields ranging from 0.01 to 0.29, and fluorescence lifetimes from 3 to 42 ns. In addition, several of these DNA-CAs exhibited white emission in aqueous solution. The molecules were used in multispectral cell imaging experiments and were taken up into cells passively. The results expand the range of emission properties that can be achieved in water with all-hydrocarbon chromophores and establish the use of the DNA scaffold to arrange carbon layers in homogeneous, rapidly synthesized assemblies. PMID:28841010

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds.

PubMed

Bhaskar, Birru; Owen, Robert; Bahmaee, Hossein; Rao, Parcha Sreenivasa; Reilly, Gwendolen C

2018-06-01

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

DNA packaging intermediates of bacteriophage Φ174

PubMed Central

Music, Cynthia L; Cheng, R Holland; Bowen, Zorina; McKenna, Robert; Rossmann, Michael G; Baker, Timothy S; Incardona, Nino L

2014-01-01

Background Like many viruses, bacteriophage ΦX174 packages its I)NA genome into a procapsid that is assembled from structural intermediates and scaffolding proteins. The procapsid contains the structural proteins F, G and H, as well as the scaffolding proteins B and D. Provirions are formed by packaging of DNA together with the small internal J proteins, while losing at least some of the B scaffolding proteins. Eventually, loss of the I) scaffolding proteins and the remaining B proteins leads to the formation of mature virions. Results ΦX174 108S 'procapsids' have been purified in milligram quantities by removing 114S (mature virion) and 70S (abortive capsid) particles from crude lysates by differential precipitation with polyethylene glycol. 132S 'provirions' were purified on sucrose gradients in the presence of EDTA. Cryo-electron microscopy (cryo-EM) was used to obtain reconstructions of procapsids and provirions. Although these are very similar to each other, their structures differ greatly from that of the virion. The F and G proteins, whose atomic structures in virions were previously determined from X-ray crystallography, were fitted into the cryo-EM reconstructions. This showed that the pentamer of G proteins on each five-fold vertex changes its conformation only slightly during DNA packaging and maturation, whereas major tertiary and quaternary structural changes occur in the F protein. The procapsids and provirions were found to contain 120 copies of the I) protein arranged as tetramers on the twofold axes. IDNA might enter procapsids through one of the 30 Å diameter holes on the icosahedral three-fold axes. Conclusions Combining cryo-EM image reconstruction and X-ray crystallography has revealed the major conformational changes that can occur in viral assembly. The function of the scaffolding proteins may be, in part, to support weak interactions between the structural proteins in the procapsids and to cover surfaces that are subsequently required for subunit–subunit interaction in the virion. The structures presented here are, therefore, analogous to chaperone proteins complexed with folding intermediates of a substrate. PMID:7613866

Regulated release of a novel non-viral gene delivery vector from electrospun coaxial fiber mesh scaffolds

NASA Astrophysics Data System (ADS)

Saraf, Anita

The development of novel strategies for tissue engineering entails the evolution of biopolymers into multifunctional constructs that can support the proliferation of cells and stimulate their differentiation into functional tissues. With that in mind, biocompatible polymers were fabricated into a novel gene delivery agent as well as three dimensional scaffolds that act as reservoirs and controlled release constructs. To fabricate a novel gene delivery agent a commercially available cationic polymer, poly(ethylenimine), PEI, was chemically conjugated to a ubiquitous glycosaminoglycan, hyaluronic acid (HA). The novel polymer, PEI-HA, had significantly reduced toxicity and improved transfection efficiency with multipotent human mesenchymal stem cells. This transfection efficiency could further be modulated by changing the concentration of sodium chloride and temperature used to assemble PEI-HA/DNA complexes. To facilitate the regulated delivery of these complexes in the context of tissue engineering, an emerging technology for scaffold fabrication, coaxial electrospinning was adapted to include PEI-HA and plasmid DNA within the scaffold fibers. Initially, a factorial design was employed to assess the influence of processing parameters in the absence of gene delivery vectors and plasmids. The study elucidated the role of sheath polymer concentration and core polymer concentration and molecular weight and the presence of sodium chloride on fiber diameters and morphologies. Subsequently, PEI-HA and plasmid DNA were entrapped within the sheath and core compartments of these fibers and the influence of processing parameters was assessed in the context of fiber diameter, release kinetics and transfection efficiency over a period of 60 days. The release of PEI-HA was found to be dependent upon the loading dose of the vector and plasmid. However, the transfection efficiency correlated to the core polymer properties, concentration and molecular weight. The processing parameters could modulate cell transfection for up to 21 days and continue to transfect cells for up to 60 days. Thus, scaffolds with tunable release kinetics and transfection efficiencies can be fabricated using coaxial electrospinning, which can further be used for tissue engineering and gene delivery applications.

An Autologous Bone Marrow Mesenchymal Stem Cell–Derived Extracellular Matrix Scaffold Applied with Bone Marrow Stimulation for Cartilage Repair

PubMed Central

Tang, Cheng; Jin, Chengzhe; Du, Xiaotao; Yan, Chao; Min, Byoung-Hyun; Xu, Yan

2014-01-01

Purpose: It is well known that implanting a bioactive scaffold into a cartilage defect site can enhance cartilage repair after bone marrow stimulation (BMS). However, most of the current scaffolds are derived from xenogenous tissue and/or artificial polymers. The implantation of these scaffolds adds risks of pathogen transmission, undesirable inflammation, and other immunological reactions, as well as ethical issues in clinical practice. The current study was undertaken to evaluate the effectiveness of implanting autologous bone marrow mesenchymal stem cell–derived extracellular matrix (aBMSC-dECM) scaffolds after BMS for cartilage repair. Methods: Full osteochondral defects were performed on the trochlear groove of both knees in 24 rabbits. One group underwent BMS only in the right knee (the BMS group), and the other group was treated by implantation of the aBMSC-dECM scaffold after BMS in the left knee (the aBMSC-dECM scaffold group). Results: Better repair of cartilage defects was observed in the aBMSC-dECM scaffold group than in the BMS group according to gross observation, histological assessments, immunohistochemistry, and chemical assay. The glycosaminoglycan and DNA content, the distribution of proteoglycan, and the distribution and arrangement of type II and I collagen fibers in the repaired tissue in the aBMSC-dECM scaffold group at 12 weeks after surgery were similar to that surrounding normal hyaline cartilage. Conclusions: Implanting aBMSC-dECM scaffolds can enhance the therapeutic effect of BMS on articular cartilage repair, and this combination treatment is a potential method for successful articular cartilage repair. PMID:24666429

Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold

NASA Astrophysics Data System (ADS)

Li, Yizhou; De Luca, Roberto; Cazzamalli, Samuele; Pretto, Francesca; Bajic, Davor; Scheuermann, Jörg; Neri, Dario

2018-03-01

In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

PubMed Central

Chainani, Abby; Hippensteel, Kirk J.; Kishan, Alysha; Garrigues, N. William; Ruch, David S.; Guilak, Farshid

2013-01-01

Full-thickness rotator cuff tears are one of the most common causes of shoulder pain in people over the age of 65. High retear rates and poor functional outcomes are common after surgical repair, and currently available extracellular matrix scaffold patches have limited abilities to enhance new tendon formation. In this regard, tissue-engineered scaffolds may provide a means to improve repair of rotator cuff tears. Electrospinning provides a versatile method for creating nanofibrous scaffolds with controlled architectures, but several challenges remain in its application to tissue engineering, such as cell infiltration through the full thickness of the scaffold as well as control of cell growth and differentiation. Previous studies have shown that ligament-derived extracellular matrix may enhance differentiation toward a tendon or ligament phenotype by human adipose stem cells (hASCs). In this study, we investigated the use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaffolds compared to fibronectin (FN) or phosphate-buffered saline (PBS) coating for use in rotator cuff tendon tissue engineering. Multilayered poly(ɛ-caprolactone) scaffolds were prepared by sequentially collecting electrospun layers onto the surface of a grounded saline solution into a single scaffold. Scaffolds were then coated with TDM, FN, or PBS and seeded with hASCs. Scaffolds were maintained without exogenous growth factors for 28 days in culture and evaluated for protein content (by immunofluorescence and biochemical assay), markers of tendon differentiation, and tensile mechanical properties. The collagen content was greatest by day 28 in TDM-scaffolds. Gene expression of type I collagen, decorin, and tenascin C increased over time, with no effect of scaffold coating. Sulfated glycosaminoglycan and dsDNA contents increased over time in culture, but there was no effect of scaffold coating. The Young's modulus did not change over time, but yield strain increased with time in culture. Histology demonstrated cell infiltration through the full thickness of all scaffolds and immunofluorescence demonstrated greater expression of type I, but not type III collagen through the full thickness of the scaffold in TDM-scaffolds compared to other treatment groups. Together, these data suggest that nonaligned multilayered electrospun scaffolds permit tenogenic differentiation by hASCs and that TDM may promote some aspects of this differentiation. PMID:23808760

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds.

PubMed

Gomes, S R; Rodrigues, G; Martins, G G; Henriques, C M R; Silva, J C

2013-04-01

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solution's properties (surface tension, electrical conductivity and viscosity) and scaffold's properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation. Copyright © 2012 Elsevier B.V. All rights reserved.

The comet assay in human biomonitoring.

PubMed

Anderson, Diana; Dhawan, Alok; Laubenthal, Julian

2013-01-01

Human biomonitoring studies aim to identify potential exposures to environmental, occupational, or lifestyle toxicants in human populations and are commonly used by public health decision makers to predict disease risk. The Comet assay measures changes in genomic stability and is one of the most reliable biomarkers to indicate early biological effects, and therefore accepted by various governmental regulatory agencies. The appeal of the Comet assay lies in its relative simplicity, rapidity, sensitivity, and economic efficiency. Furthermore, the assay is known for its broad versatility, as it can be applied to virtually any human cell and easily adapted in order to detect particular biomarkers of interest, such as DNA repair capacity or single- and double-strand breaks. In a standard experiment, isolated single cells are first embedded in agarose, and then lysed in high-salt solutions in order to remove all cellular contents except the DNA attached to a nuclear scaffold. Subsequent electrophoresis results in accumulation of undamaged DNA sequences at the proximity of the nuclear scaffold, while damaged sequences migrate towards the anode. When visualized with fluorochromes, these migrated DNA fragments resemble a comet tail and can be quantified for their intensity and shape according to internationally drafted guidelines.

Generation of a scaffold free cartilage-like implant from a small amount of starting material.

PubMed

Stoddart, M J; Ettinger, L; Häuselmann, H J

2006-01-01

An autologous cellular based treatment of a traumatic cartilage injury requires a procedure whereby a biopsy of healthy cartilage is removed from the patient and the cells isolated and expanded by monolayer passage. This increases the cell number to required levels but also leads to a de-differentiation of the cells. We aim to produce a scaffold-free, de-novo implant from a biopsy of cartilage. Bovine chondrocytes were isolated from a small biopsy and expanded. The chondrocytic phenotype of the monolayer expanded cells was recovered during a period of culture in alginate and the effect of factors such as IGF1, TFGbeta1 and dexamethasone was investigated. During the alginate culture period a pre-treatment with IGF1 and dexamethasone was shown to have little effect. IGF1 however increased the glycosaminoglycan/DNA (GAG/DNA) content on day 14 to 84.95+/-5 ng/ng compared with 37.3+/-1.8 ng/ng in the controls (P<0.001). 35S labeling demonstrated an increased GAG synthesis in the presence of IGF1 (P<0.001). IGF1 also induced a increase of DNA content 1383+/-314 ng/bead compared to 512+/-19 ng/bead in the controls (P<0.001). The cells were released from the alginate and cultured in a silicon mould for a further 14 days to obtain a three dimensional implant. Releasing the cells from the alginate and casting in a mould produced an implant of defined shape which contained no foreign material. After 31 days of culture the implants contained 152.4+/-13.14 ng/ng GAG/DNA and 42.93+/-10.23 ng/ng collagen II. We believe alginate released chondrocytes provide a real alternative to artificial scaffolds.

Direct Spectroscopic Study of Reconstituted Transcription Complexes Reveals That Intrinsic Termination Is Driven Primarily by Thermodynamic Destabilization of the Nucleic Acid Framework*S

PubMed Central

Datta, Kausiki; von Hippel, Peter H.

2008-01-01

Changes in near UV circular dichroism (CD) and fluorescence spectra of site-specifically placed pairs of 2-aminopurine residues have been used to probe the roles of the RNA hairpin and the RNA-DNA hybrid in controlling intrinsic termination of transcription. Functional transcription complexes were assembled directly by mixing preformed nucleic acid scaffolds of defined sequence with T7 RNA polymerase (RNAP). Scaffolds containing RNA hairpins immediately upstream of a GC-rich hybrid formed complexes of reduced stability, whereas the same hairpins adjacent to a hybrid of rU-dA base pairs triggered complex dissociation and transcript release. 2-Aminopurine probes at the upstream ends of the hairpin stems show that the hairpins open on RNAP binding and that stem re-formation begins after one or two RNA bases on the downstream side of the stem have emerged from the RNAP exit tunnel. Hairpins directly adjacent to the RNA-DNA hybrid weaken RNAP binding, decrease elongation efficiency, and disrupt the upstream end of the hybrid as well as interfere with the movement of the template base at the RNAP active site. Probing the edges of the DNA transcription bubble demonstrates that termination hairpins prevent translocation of the RNAP, suggesting that they transiently “lock” the polymerase to the nucleic acid scaffold and, thus, hold the RNA-DNA hybrid “in frame.” At intrinsic terminators the weak rU-dA hybrid and the adjacent termination hairpin combine to destabilize the elongation complex sufficiently to permit significant transcript release, whereas hairpin-dependent pausing provides time for the process to go to completion. PMID:18070878

The in vitro and in vivo biocompatibility evaluation of electrospun recombinant spider silk protein/PCL/gelatin for small caliber vascular tissue engineering scaffolds.

PubMed

Xiang, Ping; Wang, Shan-Shan; He, Meng; Han, Yong-He; Zhou, Zhi-Hua; Chen, Deng-Long; Li, Min; Ma, Lena Q

2018-03-01

Recombinant spider silk protein (pNSR32) and gelatin (Gt) were demonstrated to enhance cytocompatibility of electrospun pNSR32/PCL/Gt scaffold. However, its potential pro-inflammatory effects and interactions with tissue and blood are unknown. In this study, the physicochemical properties and in vitro and in vivo biocompatibility of such scaffolds were evaluated. The results showed that the pNSR32/PCL/Gt scaffold possessed larger average fiber diameters, wider fiber diameter distribution and faster degradation rate than that of pNSR32/PCL and PCL scaffolds. The addition of pNSR32 and Gt had little influence on the hemolysis and plasma re-calcification time, but prolonged kinetic clotting time and reduced the platelet adhesion. The Il-6 and Tnf-α mRNA expression levels were up-regulated in macrophages seeded on the PCL and pNSR32/PCL scaffolds. The lowest release of IL-6 and TNF-α appeared in the pNSR32/PCL/Gt scaffold. Histological results revealed that the PCL and pNSR32/PCL scaffolds elicited severe host tissue responses after implantation, while prominent ingrowth of host cells were observed in the pNSR32/PCL and pNSR32/PCL/Gt scaffolds. The comet assay and bone marrow micronucleus test demonstrated that the pNSR32/PCL/Gt scaffold did not increase the frequency of DNA damage or bone marrow micronucleus. In short, this study confirmed that the pNSR32/PCL/Gt scaffold exhibited better blood and tissue compatibility than pNSR32/PCL and PCL scaffolds. No induction of genotoxicity and inflammatory factor releases makes the pNSR32/PCL/Gt scaffold a good candidate for engineering small diameter vascular tissue. Copyright © 2017. Published by Elsevier B.V.

Scaffold protein enigma homolog 1 overcomes the repression of myogenesis activation by inhibitor of DNA binding 2

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

Nakatani, Miyuki; Ito, Jumpei; Japan Society for the Promotion of Science, Tokyo, 102-0083

Enigma Homolog 1 (ENH1) is a scaffold protein for signaling proteins and transcription factors. Previously, we reported that ENH1 overexpression promotes the differentiation of C2C12 myoblasts. However, the molecular mechanism underlying the role of ENH1 in the C2C12 cells differentiation remains elusive. ENH1 was shown to inhibit the proliferation of neuroblastoma cells by sequestering Inhibitor of DNA binding protein 2 (Id2) in the cytosol. Id2 is a repressor of basic Helix-Loop-Helix transcription factors activity and prevents myogenesis. Here, we found that ENH1 overcome the Id2 repression of C2C12 cells myogenic differentiation and that ENH1 overexpression promotes mice satellite cells activation, the firstmore » step toward myogenic differentiation. In addition, we show that ENH1 interacted with Id2 in C2C12 cells and mice satellite cells. Collectively, our results suggest that ENH1 plays an important role in the activation of myogenesis through the repression of Id2 activity. -- Highlights: •Enigma Homolog 1 (ENH1) is a scaffold protein. •ENH1 binds to inhibitor of DNA binding 2 (Id2) in myoblasts. •ENH1 overexpression overcomes the Id2's repression of myogenesis. •The Id2-ENH1 complex play an important role in the activation of myogenesis.« less

Effect of recombinant galectin-1 on the growth of immortal rat chondrocyte on chitosan-coated PLGA scaffold.

PubMed

Chen, Shiang-Jiuun; Lin, Chien-Chung; Tuan, Wei-Cheh; Tseng, Ching-Shiow; Huang, Rong-Nan

2010-06-15

The effect of galectin-1 (GAL1) on the growth of immortal rat chondrocyte (IRC) on chitosan-modified PLGA scaffold is investigated. The experimental results showed that water absorption ratio of chitosan-modified PLGA scaffold was 70% higher than that of PLGA alone after immersion in ddH(2)O for 2 weeks, indicating that chitosan-modification significantly enhances the hydrophilicity of PLGA. The experimental results also showed that GALl efficiently and spontaneously coats the chitosan-PLGA scaffold surface to promote adhesion and growth of immortal rat chondrocyte (IRC). To investigate the effect of endogenous GAL1, the full-length GAL1 cDNAs were cloned and constructed into pcDNA3.1 vectors to generate a plasmid expressed in IRC (IRC-GAL1). The results showed that IRC-GAL1 growth was significantly higher than that of IRC on chitosan-PLGA scaffold. The GAL1-potentiated IRC growth on chitosan-PLGA scaffold was dose-dependently inhibited by TDG (specific inhibitor of GAL1 binding). These results strongly suggest that GAL1 is critical for enhancing IRC cell adhesion and growth on chitosan-PLGA scaffold. Moreover, GAL1-coating or expression tends to promote IRC cell-cell aggregation on chitosan-PLGA scaffold and significantly enhances IRC migration. These results suggest that GAL1 probably could induce tissue differentiation and facilitates cartilage reconstruction. In conclusion, the experimental results suggest that both GAL1 and chitosan are important for enhancing IRC cell adhesion and growth on PLGA scaffold, and GAL1 is a potential biomaterial for tissue engineering. (c) 2009 Wiley Periodicals, Inc.

Enhancer of rudimentary homologue interacts with scaffold attachment factor B at the nuclear matrix to regulate SR protein phosphorylation.

PubMed

Drakouli, Sotiria; Lyberopoulou, Aggeliki; Papathanassiou, Maria; Mylonis, Ilias; Georgatsou, Eleni

2017-08-01

Scaffold attachment factor B1 (SAFB1) is an integral component of the nuclear matrix of vertebrate cells. It binds to DNA on scaffold/matrix attachment region elements, as well as to RNA and a multitude of different proteins, affecting basic cellular activities such as transcription, splicing and DNA damage repair. In the present study, we show that enhancer of rudimentary homologue (ERH) is a new molecular partner of SAFB1 and its 70% homologous paralogue, scaffold attachment factor B2 (SAFB2). ERH interacts directly in the nucleus with the C-terminal Arg-Gly-rich region of SAFB1/2 and co-localizes with it in the insoluble nuclear fraction. ERH, a small ubiquitous protein with striking homology among species and a unique structure, has also been implicated in fundamental cellular mechanisms. Our functional analyses suggest that the SAFB/ERH interaction does not affect SAFB1/2 function in transcription (e.g. as oestrogen receptor α co-repressors), although it reverses the inhibition exerted by SAFB1/2 on the splicing kinase SR protein kinase 1 (SRPK1), which also binds on the C-terminus of SAFB1/2. Accordingly, ERH silencing decreases lamin B receptor and SR protein phosphorylation, which are major SRPK1 substrates, further substantiating the role of SAFB1 and SAFB2 in the co-ordination of nuclear function. © 2017 Federation of European Biochemical Societies.

Biofunctional Ionic-Doped Calcium Phosphates: Silk Fibroin Composites for Bone Tissue Engineering Scaffolding.

PubMed

Pina, S; Canadas, R F; Jiménez, G; Perán, M; Marchal, J A; Reis, R L; Oliveira, J M

2017-01-01

The treatment and regeneration of bone defects caused by traumatism or diseases have not been completely addressed by current therapies. Lately, advanced tools and technologies have been successfully developed for bone tissue regeneration. Functional scaffolding materials such as biopolymers and bioresorbable fillers have gained particular attention, owing to their ability to promote cell adhesion, proliferation, and extracellular matrix production, which promote new bone growth. Here, we present novel biofunctional scaffolds for bone regeneration composed of silk fibroin (SF) and β-tricalcium phosphate (β-TCP) and incorporating Sr, Zn, and Mn, which were successfully developed using salt-leaching followed by a freeze-drying technique. The scaffolds presented a suitable pore size, porosity, and high interconnectivity, adequate for promoting cell attachment and proliferation. The degradation behavior and compressive mechanical strengths showed that SF/ionic-doped TCP scaffolds exhibit improved characteristics for bone tissue engineering when compared with SF scaffolds alone. The in vitro bioactivity assays using a simulated body fluid showed the growth of an apatite layer. Furthermore, in vitro assays using human adipose-derived stem cells presented different effects on cell proliferation/differentiation when varying the doping agents in the biofunctional scaffolds. The incorporation of Zn into the scaffolds led to improved proliferation, while the Sr- and Mn-doped scaffolds presented higher osteogenic potential as demonstrated by DNA quantification and alkaline phosphatase activity. The combination of Sr with Zn led to an influence on cell proliferation and osteogenesis when compared with single ions. Our results indicate that biofunctional ionic-doped composite scaffolds are good candidates for further in vivo studies on bone tissue regeneration. © 2017 S. Karger AG, Basel.

Development and Characterization of Acellular Extracellular Matrix Scaffolds from Porcine Menisci for Use in Cartilage Tissue Engineering

PubMed Central

Chen, Ying-Chen; Chen, Ray-Neng; Jhan, Hua-Jing; Liu, Der-Zen; Ho, Hsiu-O; Mao, Yong; Kohn, Joachim

2015-01-01

Given the growing number of arthritis patients and the limitations of current treatments, there is great urgency to explore cartilage substitutes by tissue engineering. In this study, we developed a novel decellularization method for menisci to prepare acellular extracellular matrix (ECM) scaffolds with minimal adverse effects on the ECM. Among all the acid treatments, formic acid treatment removed most of the cellular contents and preserved the highest ECM contents in the decellularized porcine menisci. Compared with fresh porcine menisci, the content of DNA decreased to 4.10%±0.03%, and there was no significant damage to glycosaminoglycan (GAG) or collagen. Histological staining also confirmed the presence of ECM and the absence of cellularity. In addition, a highly hydrophilic scaffold with three-dimensional interconnected porous structure was fabricated from decellularized menisci tissue. Human chondrocytes showed enhanced cell proliferation and synthesis of chondrocyte ECM including type II collagen and GAG when cultured in this acellular scaffold. Moreover, the scaffold effectively supported chondrogenesis of human bone marrow-derived mesenchymal stem cells. Finally, in vivo implantation was conducted in rats to assess the biocompatibility of the scaffolds. No significant inflammatory response was observed. The acellular ECM scaffold provided a native environment for cells with diverse physiological functions to promote cell proliferation and new tissue formation. This study reported a novel way to prepare decellularized meniscus tissue and demonstrated the potential as scaffolds to support cartilage repair. PMID:25919905

Improving effects of chitosan nanofiber scaffolds on osteoblast proliferation and maturation

PubMed Central

Ho, Ming-Hua; Liao, Mei-Hsiu; Lin, Yi-Ling; Lai, Chien-Hao; Lin, Pei-I; Chen, Ruei-Ming

2014-01-01

Osteoblast maturation plays a key role in regulating osteogenesis. Electrospun nanofibrous products were reported to possess a high surface area and porosity. In this study, we developed chitosan nanofibers and examined the effects of nanofibrous scaffolds on osteoblast maturation and the possible mechanisms. Macro- and micro observations of the chitosan nanofibers revealed that these nanoproducts had a flat surface and well-distributed fibers with nanoscale diameters. Mouse osteoblasts were able to attach onto the chitosan nanofiber scaffolds, and the scaffolds degraded in a time-dependent manner. Analysis by scanning electron microscopy further showed mouse osteoblasts adhered onto the scaffolds along the nanofibers, and cell–cell communication was also detected. Mouse osteoblasts grew much better on chitosan nanofiber scaffolds than on chitosan films. In addition, human osteoblasts were able to adhere and grow on the chitosan nanofiber scaffolds. Interestingly, culturing human osteoblasts on chitosan nanofiber scaffolds time-dependently increased DNA replication and cell proliferation. In parallel, administration of human osteoblasts onto chitosan nanofibers significantly induced osteopontin, osteocalcin, and alkaline phosphatase (ALP) messenger (m)RNA expression. As to the mechanism, chitosan nanofibers triggered runt-related transcription factor 2 mRNA and protein syntheses. Consequently, results of ALP-, alizarin red-, and von Kossa-staining analyses showed that chitosan nanofibers improved osteoblast mineralization. Taken together, results of this study demonstrate that chitosan nanofibers can stimulate osteoblast proliferation and maturation via runt-related transcription factor 2-mediated regulation of osteoblast-associated osteopontin, osteocalcin, and ALP gene expression. PMID:25246786

Electrochemical Aptamer Scaffold Biosensors for Detection of Botulism and Ricin Proteins.

PubMed

Daniel, Jessica; Fetter, Lisa; Jett, Susan; Rowland, Teisha J; Bonham, Andrew J

2017-01-01

Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of molecular targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins. Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin. This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

Scaffold Functions of 14-3-3 Adaptors in B Cell Immunoglobulin Class Switch DNA Recombination

PubMed Central

White, Clayton A.; Li, Guideng; Pone, Egest J.; Xu, Zhenming; Casali, Paolo

2013-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5′-AGCT-3′ repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S–S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180–198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR. PMID:24282540

A composite scaffold of MSC affinity peptide-modified demineralized bone matrix particles and chitosan hydrogel for cartilage regeneration

NASA Astrophysics Data System (ADS)

Meng, Qingyang; Man, Zhentao; Dai, Linghui; Huang, Hongjie; Zhang, Xin; Hu, Xiaoqing; Shao, Zhenxing; Zhu, Jingxian; Zhang, Jiying; Fu, Xin; Duan, Xiaoning; Ao, Yingfang

2015-12-01

Articular cartilage injury is still a significant challenge because of the poor intrinsic healing potential of cartilage. Stem cell-based tissue engineering is a promising technique for cartilage repair. As cartilage defects are usually irregular in clinical settings, scaffolds with moldability that can fill any shape of cartilage defects and closely integrate with the host cartilage are desirable. In this study, we constructed a composite scaffold combining mesenchymal stem cells (MSCs) E7 affinity peptide-modified demineralized bone matrix (DBM) particles and chitosan (CS) hydrogel for cartilage engineering. This solid-supported composite scaffold exhibited appropriate porosity, which provided a 3D microenvironment that supports cell adhesion and proliferation. Cell proliferation and DNA content analysis indicated that the DBM-E7/CS scaffold promoted better rat bone marrow-derived MSCs (BMMSCs) survival than the CS or DBM/CS groups. Meanwhile, the DBM-E7/CS scaffold increased matrix production and improved chondrogenic differentiation ability of BMMSCs in vitro. Furthermore, after implantation in vivo for four weeks, compared to those in control groups, the regenerated issue in the DBM-E7/CS group exhibited translucent and superior cartilage-like structures, as indicated by gross observation, histological examination, and assessment of matrix staining. Overall, the functional composite scaffold of DBM-E7/CS is a promising option for repairing irregularly shaped cartilage defects.

Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications.

PubMed

Lange, P; Greco, K; Partington, L; Carvalho, C; Oliani, S; Birchall, M A; Sibbons, P D; Lowdell, M W; Ansari, T

2017-03-01

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no-vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p<0.05.). Our approach did not impact on the collagen or glycosaminoglycan content or on the biomechanical properties of the scaffolds. We applied the vacuum technology to human tracheae, which, when implanted in vivo showed no significant adverse immunological response. The addition of a vacuum to a conventional decellularization protocol significantly reduces production time, whilst providing a suitable scaffold. This increases clinical utility and lowers production costs. To our knowledge this is the first time that vacuum assisted decellularization has been explored. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

PubMed

Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

2015-11-17

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection

PubMed Central

Bantele, Susanne CS; Ferreira, Pedro; Gritenaite, Dalia; Boos, Dominik; Pfander, Boris

2017-01-01

DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here, we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Budding yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11/TOPBP1, respectively. In yeast, this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin, and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice. DOI: http://dx.doi.org/10.7554/eLife.21687.001 PMID:28063255

Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

NASA Astrophysics Data System (ADS)

Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

2017-06-01

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

Nanocomposite scaffold fabrication by incorporating gold nanoparticles into biodegradable polymer matrix: Synthesis, characterization, and photothermal effect.

PubMed

Abdelrasoul, Gaser N; Farkas, Balazs; Romano, Ilaria; Diaspro, Alberto; Beke, Szabolcs

2015-11-01

Nanoparticle incorporation into scaffold materials is a valuable route to deliver various therapeutic agents, such as drug molecules or large biomolecules, proteins (e.g. DNA or RNA) into their targets. In particular, gold nanoparticles (Au NPs) with their low inherent toxicity, tunable stability and high surface area provide unique attributes facilitating new delivery strategies. A biodegradable, photocurable polymer resin, polypropylene fumarate (PPF) along with Au NPs were utilized to synthesize a hybrid nanocomposite resin, directly exploitable in stereolithography (SL) processes. To increase the particles' colloidal stability, the Au NP nanofillers were coated with polyvinyl pyrrolidone (PVP). The resulting resin was used to fabricate a new type of composite scaffold via mask projection excimer laser stereolithography. The thermal properties of the nanocomposite scaffolds were found to be sensitive to the concentration of NPs. The mechanical properties were augmented by the NPs up to 0.16μM, though further increase in the concentration led to a gradual decrease. Au NP incorporation rendered the biopolymer scaffolds photosensitive, i.e. the presence of Au NPs enhanced the optical absorption of the scaffolds as well, leading to possible localized temperature rise when irradiated with 532nm laser, known as the photothermal effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Photoinduced silver nanoparticles/nanorings on plasmid DNA scaffolds.

PubMed

Liu, Jianhua; Zhang, Xiaoliang; Yu, Mei; Li, Songmei; Zhang, Jindan

2012-01-23

Biological scaffolds are being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. Toroidal plasmid DNA separated from the Bacillus host is applied as a sacrificial mold for the synthesis of silver nanoparticles and nanorings. The photoirradiation method is applied to reduce Ag(I) on the plasmid. The nanoparticles are obtained by varying the concentration of the Ag(I) ion solution and the exposure time of the plasmid-Ag(I) complex under UV light at 254 nm and room temperature. It is found that the plasmid serves not only as a template but also as a reductant to drive the silver nucleation and deposition. The resulting nanoparticles have a face-centered cubic (fcc) crystal structure and 20-30 nm average diameter. The detailed mechanism is discussed, and other metals or alloys could also be synthesized with this method. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure and conformational dynamics of scaffolded DNA origami nanoparticles

DTIC Science & Technology

2017-05-08

all-atom molecular dynamics and coarse-grained finite element modeling to DX-based nanoparticles to elucidate their fine-scale and global conforma... finite element (FE) modeling approach CanDo is also routinely used to predict the 3D equilibrium conformation of programmed DNA assemblies based on a...model with both experimental cryo-electron microscopy (cryo-EM) data and all-atom modeling. MATERIALS AND METHODS Lattice-free finite element model

Multilayer DNA origami packed on hexagonal and hybrid lattices.

PubMed

Ke, Yonggang; Voigt, Niels V; Gothelf, Kurt V; Shih, William M

2012-01-25

"Scaffolded DNA origami" has been proven to be a powerful and efficient approach to construct two-dimensional or three-dimensional objects with great complexity. Multilayer DNA origami has been demonstrated with helices packing along either honeycomb-lattice geometry or square-lattice geometry. Here we report successful folding of multilayer DNA origami with helices arranged on a close-packed hexagonal lattice. This arrangement yields a higher density of helical packing and therefore higher resolution of spatial addressing than has been shown previously. We also demonstrate hybrid multilayer DNA origami with honeycomb-lattice, square-lattice, and hexagonal-lattice packing of helices all in one design. The availability of hexagonal close-packing of helices extends our ability to build complex structures using DNA nanotechnology. © 2011 American Chemical Society

DNA Brick Crystals with Prescribed Depth

PubMed Central

Ke, Yonggang; Ong, Luvena L.; Sun, Wei; Song, Jie; Dong, Mingdong; Shih, William M.; Yin, Peng

2014-01-01

We describe a general framework for constructing two-dimensional crystals with prescribed depth and sophisticated three-dimensional features. These crystals may serve as scaffolds for the precise spatial arrangements of functional materials for diverse applications. The crystals are self-assembled from single-stranded DNA components called DNA bricks. We demonstrate the experimental construction of DNA brick crystals that can grow to micron-size in the lateral dimensions with precisely controlled depth up to 80 nanometers. They can be designed to display user-specified sophisticated three-dimensional nanoscale features, such as continuous or discontinuous cavities and channels, and to pack DNA helices at parallel and perpendicular angles relative to the plane of the crystals. PMID:25343605

A label-free fluorescent direct detection of live Salmonella typhimurium using cascade triple trigger sequences-regenerated strand displacement amplification and hairpin template-generated-scaffolded silver nanoclusters.

PubMed

Zhang, Peng; Liu, Hui; Li, Xiaocheng; Ma, Suzhen; Men, Shuai; Wei, Heng; Cui, Jingjing; Wang, Hongning

2017-01-15

The harm of Salmonella typhimurium (S. typhimurium) to public health mainly by the consumption of contaminated agricultural products or water stresses an urgent need for rapid detection methods to help control the spread of S. typhimurium. In this work, an intelligently designed sensor system took creative advantage of triple trigger sequences-regenerated strand displacement amplification and self-protective hairpin template-generated-scaffolded silver nanoclusters (AgNCs) for the first time. In the presence of live S. typhimurium, single-stranded trigger sequences were released from aptamer-trigger sequences complex, initiating a branch migration to open the hairpin template I containing complementary scaffolds of AgNCs. Then the first strand displacement amplification was induced to produce numerous scaffolds of AgNCs and reporter strands which initiated a branch migration to open the hairpin template II containing complementary scaffolds of AgNCs. Then the second strand displacement amplification was induced to generate numerous scaffolds of AgNCs and trigger sequences which initiated the third branch migration and strand displacement amplification to produce numerous scaffolds of AgNCs and reporter strands in succession. Cyclically, the reproduction of the trigger sequences and cascade successive production of scaffolds were achieved successfully, forming highly fluorescent AgNCs, thus providing significantly enhanced fluorescent signals to achieve ultrasensitive detection of live S. typhimurium down to 50 CFU/mL with a linear range from 10 2 to 10 7 CFU/mL. It is the first report on a fluorescent biosensor for detecting viable S. typhimurium directly, which can distinguish from heat denatured S. typhimurium. And it develops a new strategy to generate the DNA-scaffolds for forming AgNCs. Copyright © 2016 Elsevier B.V. All rights reserved.

3D printed hyperelastic "bone" scaffolds and regional gene therapy: A novel approach to bone healing.

PubMed

Alluri, Ram; Jakus, Adam; Bougioukli, Sofia; Pannell, William; Sugiyama, Osamu; Tang, Amy; Shah, Ramille; Lieberman, Jay R

2018-04-01

The purpose of this study was to evaluate the viability of human adipose-derived stem cells (ADSCs) transduced with a lentiviral (LV) vector to overexpress bone morphogenetic protein-2 (BMP-2) loaded onto a novel 3D printed scaffold. Human ADSCs were transduced with a LV vector carrying the cDNA for BMP-2. The transduced cells were loaded onto a 3D printed Hyperelastic "Bone" (HB) scaffold. In vitro BMP-2 production was assessed using enzyme-linked immunosorbent assay analysis. The ability of ADSCs loaded on the HB scaffold to induce in vivo bone formation in a hind limb muscle pouch model was assessed in the following groups: ADSCs transduced with LV-BMP-2, LV-green fluorescent protein, ADSCs alone, and empty HB scaffolds. Bone formation was assessed using radiographs, histology and histomorphometry. Transduced ADSCs BMP-2 production on the HB scaffold at 24 hours was similar on 3D printed HB scaffolds versus control wells with transduced cells alone, and continued to increase after 1 and 2 weeks of culture. Bone formation was noted in LV-BMP-2 animals on plain radiographs at 2 and 4 weeks after implantation; no bone formation was noted in the other groups. Histology demonstrated that the LV-BMP-2 group was the only group that formed woven bone and the mean bone area/tissue area was significantly greater when compared with the other groups. 3D printed HB scaffolds are effective carriers for transduced ADSCs to promote bone repair. The combination of gene therapy and tissue engineered scaffolds is a promising multidisciplinary approach to bone repair with significant clinical potential. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1104-1110, 2018. © 2018 Wiley Periodicals, Inc.

Tissue engineering of heart valves: in vitro experiences.

PubMed

Sodian, R; Hoerstrup, S P; Sperling, J S; Daebritz, S H; Martin, D P; Schoen, F J; Vacanti, J P; Mayer, J E

2000-07-01

Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

Nanofibers and their applications in tissue engineering

PubMed Central

Vasita, Rajesh; Katti, Dhirendra S

2006-01-01

Developing scaffolds that mimic the architecture of tissue at the nanoscale is one of the major challenges in the field of tissue engineering. The development of nanofibers has greatly enhanced the scope for fabricating scaffolds that can potentially meet this challenge. Currently, there are three techniques available for the synthesis of nanofibers: electrospinning, self-assembly, and phase separation. Of these techniques, electrospinning is the most widely studied technique and has also demonstrated the most promising results in terms of tissue engineering applications. The availability of a wide range of natural and synthetic biomaterials has broadened the scope for development of nanofibrous scaffolds, especially using the electrospinning technique. The three dimensional synthetic biodegradable scaffolds designed using nanofibers serve as an excellent framework for cell adhesion, proliferation, and differentiation. Therefore, nanofibers, irrespective of their method of synthesis, have been used as scaffolds for musculoskeletal tissue engineering (including bone, cartilage, ligament, and skeletal muscle), skin tissue engineering, vascular tissue engineering, neural tissue engineering, and as carriers for the controlled delivery of drugs, proteins, and DNA. This review summarizes the currently available techniques for nanofiber synthesis and discusses the use of nanofibers in tissue engineering and drug delivery applications. PMID:17722259

Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering.

PubMed

Duisit, Jérôme; Amiel, Hadrien; Wüthrich, Tsering; Taddeo, Adriano; Dedriche, Adeline; Destoop, Vincent; Pardoen, Thomas; Bouzin, Caroline; Joris, Virginie; Magee, Derek; Vögelin, Esther; Harriman, David; Dessy, Chantal; Orlando, Giuseppe; Behets, Catherine; Rieben, Robert; Gianello, Pierre; Lengelé, Benoît

2018-06-01

Human ear reconstruction is recognized as the emblematic enterprise in tissue engineering. Up to now, it has failed to reach human applications requiring appropriate tissue complexity along with an accessible vascular tree. We hereby propose a new method to process human auricles in order to provide a poorly immunogenic, complex and vascularized ear graft scaffold. 12 human ears with their vascular pedicles were procured. Perfusion-decellularization was applied using a SDS/polar solvent protocol. Cell and antigen removal was examined by histology and DNA was quantified. Preservation of the extracellular matrix (ECM) was assessed by conventional and 3D-histology, proteins and cytokines quantifications. Biocompatibility was assessed by implantation in rats for up to 60 days. Adipose-derived stem cells seeding was conducted on scaffold samples and with human aortic endothelial cells whole graft seeding in a perfusion-bioreactor. Histology confirmed cell and antigen clearance. DNA reduction was 97.3%. ECM structure and composition were preserved. Implanted scaffolds were tolerated in vivo, with acceptable inflammation, remodeling, and anti-donor antibody formation. Seeding experiments demonstrated cell engraftment and viability. Vascularized and complex auricular scaffolds can be obtained from human source to provide a platform for further functional auricular tissue engineered constructs, hence providing an ideal road to the vascularized composite tissue engineering approach. The ear is emblematic in the biofabrication of tissues and organs. Current regenerative medicine strategies, with matrix from donor tissues or 3D-printed, didn't reach any application for reconstruction, because critically missing a vascular tree for perfusion and transplantation. We previously described the production of vascularized and cell-compatible scaffolds, from porcine ear grafts. In this study, we ---- applied findings directly to human auricles harvested from postmortem donors, providing a perfusable matrix that retains the ear's original complexity and hosts new viable cells after seeding. This approach unlocks the ability to achieve an auricular tissue engineering approach, associated with possible clinical translation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Enhanced biocompatibility and osteogenic potential of mesoporous magnesium silicate/polycaprolactone/wheat protein composite scaffolds.

PubMed

Kang, Yun Gyeong; Wei, Jie; Shin, Ji Won; Wu, Yan Ru; Su, Jiacan; Park, Young Shik; Shin, Jung-Woog

2018-01-01

Successful bone tissue engineering using scaffolds is primarily dependent on the properties of the scaffold, including biocompatibility, highly interconnected porosity, and mechanical integrity. In this study, we propose new composite scaffolds consisting of mesoporous magnesium silicate (m_MS), polycaprolactone (PCL), and wheat protein (WP) manufactured by a rapid prototyping technique to provide a micro/macro porous structure. Experimental groups were set based on the component ratio: (1) WP0% (m_MS:PCL:WP =30:70:0 weight per weight; w/w); (2) WP15% (m_MS:PCL:WP =30:55:15 w/w); (3) WP30% (m_MS:PCL:WP =30:40:30 w/w). Evaluation of the properties of fabricated scaffolds indicated that increasing the amount of WP improved the surface hydrophilicity and biodegradability of m_MS/PCL/WP composites, while reducing the mechanical strength. Moreover, experiments were performed to confirm the biocompatibility and osteogenic differentiation of human mesenchymal stem cells (MSCs) according to the component ratio of the scaffold. The results confirmed that the content of WP affects proliferation and osteogenic differentiation of MSCs. Based on the last day of the experiment, ie, the 14th day, the proliferation based on the amount of DNA was the best in the WP30% group, but all of the markers measured by PCR were the most expressed in the WP15% group. These results suggest that the m_MS/PCL/WP composite is a promising candidate for use as a scaffold in cell-based bone regeneration.

Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2′-deoxyuridine

PubMed Central

Tagawa, Miho; Shohda, Koh-ichiroh; Fujimoto, Kenzo; Sugawara, Tadashi; Suyama, Akira

2007-01-01

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology. PMID:17982178

DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

NASA Astrophysics Data System (ADS)

Franch, Oskar; Iacovelli, Federico; Falconi, Mattia; Juul, Sissel; Ottaviani, Alessio; Benvenuti, Claudia; Biocca, Silvia; Ho, Yi-Ping; Knudsen, Birgitta R.; Desideri, Alessandro

2016-07-01

In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01806h

Effects of 3-dimensional culture conditions (collagen-chitosan nano-scaffolds) on maturation of dendritic cells and their capacity to interact with T-lymphocytes.

PubMed

Daneshmandi, Saeed; Dibazar, Shaghayegh Pishkhan; Fateh, Shirin

2016-01-01

In the body, there is a natural three-dimensional (3D) microenvironment in which immune cells, including dendritic cells (DC), play their functions. This study evaluated the impact of using collagen-chitosan 3D nano-scaffolds in comparisons to routine 2D culture plates on DC phenotype and functions. Bone marrow-derived DC were cultured on scaffolds and plates and then stimulated with lipopolysaccharide (LPS) or chitosan-based nanoparticles (NP) for 24 h. Thereafter, DC viability, expression of maturation markers and levels of cytokines secretion were evaluated. In another set of studies, the DC were co-cultured with allogenic T-lymphocytes in both the 2D and 3D systems and effects on DC-induction of T-lymphocyte proliferation and cytokine release were analyzed. The results indicated that CD40, CD86 and MHC II marker expression and interleukin (IL)-12, IL-6 and tumor necrosis factor (TNF)-α secretion by DC were enhanced in 3D cultures in comparison to by cells maintained in the 2D states. The data also showed that DNA/chitosan NP activated DC more than LPS in the 3D system. T-Lymphocyte proliferation was induced to a greater extent by DNA/NP-treated DC when both cell types were maintained on the scaffolds. Interestingly, while DC induction of T-lymphocyte interferon (IFN)-γ and IL-4 release was enhanced in the 3D system (relative to controls), there was a suppression of transforming growth factor (TGF)-β production; effects on IL-10 secretion were variable. The results here suggested that collagen-chitosan scaffolds could provide a pro-inflammatory and activator environment to perform studies to analyze effects of exogenous agents on the induction of DC maturation, NP uptake and/or cytokines release, as well as for the ability of these cells to potentially interact with other immune system cells in vitro.

A New Protein Architecture for Processing Alkylation Damaged DNA: The Crystal Structure of DNA Glycosylase AlkD

PubMed Central

Rubinson, Emily H.; Metz, Audrey H.; O'Quin, Jami; Eichman, Brandt F.

2013-01-01

Summary DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases. PMID:18585735

DNA-repair scaffolds dampen checkpoint signalling by counteracting the adaptor Rad9.

PubMed

Ohouo, Patrice Y; Bastos de Oliveira, Francisco M; Liu, Yi; Ma, Chu Jian; Smolka, Marcus B

2013-01-03

In response to genotoxic stress, a transient arrest in cell-cycle progression enforced by the DNA-damage checkpoint (DDC) signalling pathway positively contributes to genome maintenance. Because hyperactivated DDC signalling can lead to a persistent and detrimental cell-cycle arrest, cells must tightly regulate the activity of the kinases involved in this pathway. Despite their importance, the mechanisms for monitoring and modulating DDC signalling are not fully understood. Here we show that the DNA-repair scaffolding proteins Slx4 and Rtt107 prevent the aberrant hyperactivation of DDC signalling by lesions that are generated during DNA replication in Saccharomyces cerevisiae. On replication stress, cells lacking Slx4 or Rtt107 show hyperactivation of the downstream DDC kinase Rad53, whereas activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phosphorylated histone H2A, two positive regulators of Rad9-dependent Rad53 activation. A decrease in DDC signalling results from hypomorphic mutations in RAD53 and H2A and rescues the hypersensitivity to replication stress of cells lacking Slx4 or Rtt107. We propose that the Slx4-Rtt107 complex modulates Rad53 activation by a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings show that DDC signalling is monitored and modulated through the direct action of DNA-repair factors.

Biological effects of functionalizing copolymer scaffolds with nanodiamond particles.

PubMed

Xing, Zhe; Pedersen, Torbjorn O; Wu, Xujun; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Kloss, Frank R; Waag, Thilo; Krueger, Anke; Steinmüller-Nethl, Doris; Mustafa, Kamal

2013-08-01

Significant evidence has indicated that poly(L-lactide)-co-(ɛ-caprolactone) [(poly(LLA-co-CL)] scaffolds could be one of the suitable candidates for bone tissue engineering. Oxygen-terminated nanodiamond particles (n-DP) were combined with poly(LLA-co-CL) and revealed to be positive for cell growth. In this study, we evaluated the influence of poly(LLA-co-CL) scaffolds modified by n-DP on attachment, proliferation, differentiation of bone marrow stromal cells (BMSCs) in vitro, and on bone formation using a sheep calvarial defect model. BMSCs were seeded on either poly(LLA-co-CL)- or n-DP-coated scaffolds and incubated for 1 h. Scanning electron microscopy (SEM) and fluorescence microscopy were used in addition to protein and DNA measurements to evaluate cellular attachment on the scaffolds. To determine the effect of n-DP on proliferation of BMSCs, cell/scaffold constructs were harvested after 3 days and evaluated by Bicinchoninic Acid (BCA) protein assay and SEM. In addition, the osteogenic differentiation of cells grown for 2 weeks on the various scaffolds and in a dynamic culture condition was evaluated by real-time RT-PCR. Unmodified and modified scaffolds were implanted into the calvaria of six-year-old sheep. The expression of collagen type I (COL I) and bone morphogenetic protein-2 (BMP-2) after 4 weeks as well as the formation of new bone after 12 and 24 weeks were analyzed by immunohistochemistry and histology. Scaffolds modified with n-DP supported increased cell attachment and the mRNA expression of osteopontin (OPN), bone sialoprotein (BSP), and BMP-2 were significantly increased after 2 weeks of culture. The BMSCs had spread well on the various scaffolds investigated after 3 days in the study with no significant difference in cell proliferation. Furthermore, the in vivo data revealed more positive staining of COL I and BMP-2 in relation to the n-DP-coated scaffolds after 4 weeks and presented more bone formation after 12 and 24 weeks. n-DP modification significantly increased cell attachment and differentiation of BMSCs on poly(LLA-co-CL) scaffolds in vitro and enhanced bone formation in vivo.

Advanced tissue engineering scaffold design for regeneration of the complex hierarchical periodontal structure.

PubMed

Costa, Pedro F; Vaquette, Cédryck; Zhang, Qiyi; Reis, Rui L; Ivanovski, Saso; Hutmacher, Dietmar W

2014-03-01

This study investigated the ability of an osteoconductive biphasic scaffold to simultaneously regenerate alveolar bone, periodontal ligament and cementum. A biphasic scaffold was built by attaching a fused deposition modelled bone compartment to a melt electrospun periodontal compartment. The bone compartment was coated with a calcium phosphate (CaP) layer for increasing osteoconductivity, seeded with osteoblasts and cultured in vitro for 6 weeks. The resulting constructs were then complemented with the placement of PDL cell sheets on the periodontal compartment, attached to a dentin block and subcutaneously implanted into athymic rats for 8 weeks. Scanning electron microscopy, X-ray diffraction, alkaline phosphatase and DNA content quantification, confocal laser microscopy, micro computerized tomography and histological analysis were employed to evaluate the scaffold's performance. The in vitro study showed that alkaline phosphatase activity was significantly increased in the CaP-coated samples and they also displayed enhanced mineralization. In the in vivo study, significantly more bone formation was observed in the coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted vascularization of the cell sheets, and periodontal attachment was achieved at the dentin interface. This work demonstrates that the combination of cell sheet technology together with an osteoconductive biphasic scaffold could be utilized to address the limitations of current periodontal regeneration techniques. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

DNA scaffold nanostructures for efficient and directional propagation of light harvesting cascades

NASA Astrophysics Data System (ADS)

Brown, Carl W.; Samanta, Anirban; Buckhout-White, Susan; Díaz, Sebastián. A.; Walper, Scott A.; Goldman, Ellen R.; Medintz, Igor L.

2017-08-01

The development of light harvesting systems for directed, efficient control of energy transfer at the biomolecular level has generated considerable interest in the past decade. Molecular fluorophores provide a straightforward mechanism for determining nanoscale distance changes through Förster resonance energy transfer (FRET), and many systems seek to build off of this simple yet powerful principle to provide additional functionality. The use of DNA-based integrated biomolecular devices offer many unique advantages towards this end. DNA itself is an excellent engineering material - it is innately biocompatible, quickly and cheaply synthesized, and complex structures can be readily designed in silico. It also provides an excellent scaffold for the precise patterning of various biomolecules. Here, we discuss the systems that have been recently developed which add to this toolbox, including nanostructural dye patterning, photonic wires, and the incorporation of alternative energy propagation modalities, such as semiconductor quantum dots (QD) and the bioluminescent protein luciferase. In particular, we explore the incorporation of luciferase into various nanostructural conformations, providing the capability to efficiently control energy flow directionality. We discuss the nature of this system, including unexpected spectral complexities, in the context of the field.

Modelling DNA origami self-assembly at the domain level.

PubMed

Dannenberg, Frits; Dunn, Katherine E; Bath, Jonathan; Kwiatkowska, Marta; Turberfield, Andrew J; Ouldridge, Thomas E

2015-10-28

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

Modelling DNA origami self-assembly at the domain level

NASA Astrophysics Data System (ADS)

Dannenberg, Frits; Dunn, Katherine E.; Bath, Jonathan; Kwiatkowska, Marta; Turberfield, Andrew J.; Ouldridge, Thomas E.

2015-10-01

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

Biocompatibility and hemocompatibility of efficiently decellularized whole porcine kidney for tissue engineering.

PubMed

Hussein, Kamal Hany; Saleh, Tarek; Ahmed, Ebtehal; Kwak, Ho-Hyun; Park, Kyung-Mee; Yang, Se-Ran; Kang, Byung-Jae; Choi, Ki-Young; Kang, Kyung-Sun; Woo, Heung-Myong

2018-07-01

Whole kidney decellularization is a promising approach in regenerative medicine for engineering a functional organ. The reaction of the potential host depends on the biocompatibility of these decellularized constructs. Despite the proven ability of decellularized kidney scaffolds to guide cell attachment and growth, little is known about biocompatibility and hemocompatibility of these scaffolds. Our aim is to prepare decellularized kidneys of a clinically relevant size and evaluate its biocompatibility and hemocompatibility. Porcine kidneys were cannulated via the renal artery, and then perfused with 0.1% sodium dodecyl sulfate solution. Hematoxylin and eosin as well as DAPI staining confirmed cellular clearance from native kidneys in addition to preservation of the microstructure. SEM confirmed the absence of any cellular content within the scaffold, which is maintained in a well-organized 3D architecture. Decellularized kidneys retained the intact renal vasculature upon examination with contrast radiography. The essential structural extracellular matrix molecules were well-preserved. Scaffolds were susceptible to enzymatic degradation upon collagenase treatment. Scaffolds showed a good hemocompatibility when exposed to porcine blood. Decellularization was efficient to remove 97.7% of DNA from native kidneys in addition to the immunogenic and pathogenic antigens. Scaffolds did not induce the human immune response in vitro. Decellularized kidneys were non-cytotoxic to pig kidney cells (PKs). PKs were able to grow and proliferate within the decellularized renal scaffolds with maintaining a higher function than cells grown as monolayers. Thus, we have developed a rapid decellularization technique for generating biocompatible kidney scaffolds that represents a step toward development of a transplantable organ. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2034-2047, 2018. © 2018 Wiley Periodicals, Inc.

An Overview of the Molecular Mechanisms of Recombinational DNA Repair

PubMed Central

Kowalczykowski, Stephen C.

2015-01-01

Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.

PubMed

Mashimo, Yasumasa; Mie, Masayasu; Kobatake, Eiry

2018-04-01

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

The genome sequence of sweet cherry (Prunus avium) for use in genomics-assisted breeding.

PubMed

Shirasawa, Kenta; Isuzugawa, Kanji; Ikenaga, Mitsunobu; Saito, Yutaro; Yamamoto, Toshiya; Hirakawa, Hideki; Isobe, Sachiko

2017-10-01

We determined the genome sequence of sweet cherry (Prunus avium) using next-generation sequencing technology. The total length of the assembled sequences was 272.4 Mb, consisting of 10,148 scaffold sequences with an N50 length of 219.6 kb. The sequences covered 77.8% of the 352.9 Mb sweet cherry genome, as estimated by k-mer analysis, and included >96.0% of the core eukaryotic genes. We predicted 43,349 complete and partial protein-encoding genes. A high-density consensus map with 2,382 loci was constructed using double-digest restriction site-associated DNA sequencing. Comparing the genetic maps of sweet cherry and peach revealed high synteny between the two genomes; thus the scaffolds were integrated into pseudomolecules using map- and synteny-based strategies. Whole-genome resequencing of six modern cultivars found 1,016,866 SNPs and 162,402 insertions/deletions, out of which 0.7% were deleterious. The sequence variants, as well as simple sequence repeats, can be used as DNA markers. The genomic information helps us to identify agronomically important genes and will accelerate genetic studies and breeding programs for sweet cherries. Further information on the genomic sequences and DNA markers is available in DBcherry (http://cherry.kazusa.or.jp (8 May 2017, date last accessed)). © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

DNA origami nanopillars as standards for three-dimensional superresolution microscopy.

PubMed

Schmied, Jürgen J; Forthmann, Carsten; Pibiri, Enrico; Lalkens, Birka; Nickels, Philipp; Liedl, Tim; Tinnefeld, Philip

2013-02-13

Nanopillars are promising nanostructures composed of various materials that bring new functionalities for applications ranging from photovoltaics to analytics. We developed DNA nanopillars with a height of 220 nm and a diameter of ~14 nm using the DNA origami technique. Modifying the base of the nanopillars with biotins allowed selective, upright, and rigid immobilization on solid substrates. With the help of site-selective dye labels, we visualized the structure and determined the orientation of the nanopillars by three-dimensional fluorescence superresolution microscopy. Because of their rigidity and nanometer-precise addressability, DNA origami nanopillars qualify as scaffold for the assembly of plasmonic devices as well as for three-dimensional superresolution standards.

Human Mitochondrial RNA Polymerase: Evaluation of the Single-Nucleotide-Addition Cycle on Synthetic RNA/DNA Scaffolds

PubMed Central

Smidansky, Eric D.; Arnold, Jamie J.; Reynolds, Shelley L.; Cameron, Craig E.

2013-01-01

The human mitochondrial RNA polymerase (h-mtRNAP) serves as both the transcriptase for expression and the primase for replication of mitochondrial DNA. As such, the enzyme is of fundamental importance to cellular energy metabolism, and defects in its function may be related to human disease states. Here we describe in vitro analysis of the h-mtRNAP kinetic mechanism for single, correct nucleotide incorporation. This was made possible by the development of efficient methods for expression and purification of h-mtRNAP using a bacterial system and by utilization of assays that rely on simple, synthetic RNA/DNA scaffolds without the need for mitochondrial transcription accessory proteins. We find that h-mtRNAP accomplishes single-nucleotide incorporation by using the same core steps, including conformational change steps before and after chemistry, that are prototypical for most types of nucleic acid polymerases. The polymerase binds to scaffolds via a two-step mechanism consisting of a fast initial-encounter step followed by a much slower isomerization that leads to catalytic competence. A substantial solvent deuterium kinetic isotope effect was observed for the forward reaction, but none was detectable for the reverse reaction, suggesting that chemistry is at least partially rate-limiting in the forward direction but not in the reverse. h-mtRNAP appears to exercise much more stringent surveillance over base than over sugar in determining the correctness of a nucleotide. The utility of developing the robust in vitro assays described here and of establishing a baseline of kinetic performance for the wild-type enzyme is that biological questions concerning h-mtRNAP may now begin to be addressed. PMID:21548588

The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion.

PubMed

Raimondi, Manuela T; Moretti, Matteo; Cioffi, Margherita; Giordano, Carmen; Boschetti, Federica; Laganà, Katia; Pietrabissa, Riccardo

Bioreactors allowing direct-perfusion of culture medium through tissue-engineered constructs may overcome diffusion limitations associated with static culturing, and may provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed on cells within scaffolds is directly dependent on scaffold microstructure and on bioreactor configuration. Aim of this study is to investigate optimal shear stress ranges and to quantitatively predict the levels of hydrodynamic shear imposed to cells during the experiments. Bovine articular chondrocytes were seeded on polyestherurethane foams and cultured for 2 weeks in a direct perfusion bioreactor designed to impose 4 different values of shear level at a single flow rate (0.5 ml/min). Computational fluid dynamics (CFD) simulations were carried out on reconstructions of the scaffold obtained from micro-computed tomography images. Biochemistry analyses for DNA and sGAG were performed, along with electron microscopy. The hydrodynamic shear induced on cells within constructs, as estimated by CFD simulations, ranged from 4.6 to 56 mPa. This 12-fold increase in the level of applied shear stress determined a 1.7-fold increase in the mean content in DNA and a 2.9-fold increase in the mean content in sGAG. In contrast, the mean sGAG/DNA ratio showed a tendency to decrease for increasing shear levels. Our results suggest that the optimal condition to favour sGAG synthesis in engineered constructs, at least at the beginning of culture, is direct perfusion at the lowest level of hydrodynamic shear. In conclusion, the presented results represent a first attempt to quantitatively correlate the imposed hydrodynamic shear level and the invoked biosynthetic response in 3D engineered chondrocyte systems.

Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: a combined gene therapy-cell transplantation approach.

PubMed

Jabbarzadeh, Ehsan; Starnes, Trevor; Khan, Yusuf M; Jiang, Tao; Wirtel, Anthony J; Deng, Meng; Lv, Qing; Nair, Lakshmi S; Doty, Steven B; Laurencin, Cato T

2008-08-12

One of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Efforts to induce vascular growth into tissue-engineered scaffolds have recently been dedicated to developing novel strategies to deliver specific biological factors that direct the recruitment of endothelial cell (EC) progenitors and their differentiation. The challenge, however, lies in orchestration of the cells, appropriate biological factors, and optimal factor doses. This study reports an approach as a step forward to resolving this dilemma by combining an ex vivo gene transfer strategy and EC transplantation. The utility of this approach was evaluated by using 3D poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for bone tissue engineering applications. Our goal was achieved by isolation and transfection of adipose-derived stromal cells (ADSCs) with adenovirus encoding the cDNA of VEGF. We demonstrated that the combination of VEGF releasing ADSCs and ECs results in marked vascular growth within PLAGA scaffolds. We thereby delineate the potential of ADSCs to promote vascular growth into biomaterials.

Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: A combined gene therapy–cell transplantation approach

PubMed Central

Jabbarzadeh, Ehsan; Starnes, Trevor; Khan, Yusuf M.; Jiang, Tao; Wirtel, Anthony J.; Deng, Meng; Lv, Qing; Nair, Lakshmi S.; Doty, Steven B.; Laurencin, Cato T.

2008-01-01

One of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Efforts to induce vascular growth into tissue-engineered scaffolds have recently been dedicated to developing novel strategies to deliver specific biological factors that direct the recruitment of endothelial cell (EC) progenitors and their differentiation. The challenge, however, lies in orchestration of the cells, appropriate biological factors, and optimal factor doses. This study reports an approach as a step forward to resolving this dilemma by combining an ex vivo gene transfer strategy and EC transplantation. The utility of this approach was evaluated by using 3D poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for bone tissue engineering applications. Our goal was achieved by isolation and transfection of adipose-derived stromal cells (ADSCs) with adenovirus encoding the cDNA of VEGF. We demonstrated that the combination of VEGF releasing ADSCs and ECs results in marked vascular growth within PLAGA scaffolds. We thereby delineate the potential of ADSCs to promote vascular growth into biomaterials. PMID:18678895

In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

PubMed

Xie, Lin; Zhang, Nan; Marsano, Anna; Vunjak-Novakovic, Gordana; Zhang, Yanru; Lopez, Mandi J

2013-12-01

Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

PubMed

Tresoldi, Claudia; Stefani, Ilaria; Ferracci, Gaia; Bertoldi, Serena; Pellegata, Alessandro F; Farè, Silvia; Mantero, Sara

2017-04-26

In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

Enhanced biocompatibility and osteogenic potential of mesoporous magnesium silicate/polycaprolactone/wheat protein composite scaffolds

PubMed Central

Kang, Yun Gyeong; Wei, Jie; Shin, Ji Won; Wu, Yan Ru; Su, Jiacan; Park, Young Shik; Shin, Jung-Woog

2018-01-01

Background Successful bone tissue engineering using scaffolds is primarily dependent on the properties of the scaffold, including biocompatibility, highly interconnected porosity, and mechanical integrity. Methods In this study, we propose new composite scaffolds consisting of mesoporous magnesium silicate (m_MS), polycaprolactone (PCL), and wheat protein (WP) manufactured by a rapid prototyping technique to provide a micro/macro porous structure. Experimental groups were set based on the component ratio: (1) WP0% (m_MS:PCL:WP =30:70:0 weight per weight; w/w); (2) WP15% (m_MS:PCL:WP =30:55:15 w/w); (3) WP30% (m_MS:PCL:WP =30:40:30 w/w). Results Evaluation of the properties of fabricated scaffolds indicated that increasing the amount of WP improved the surface hydrophilicity and biodegradability of m_MS/PCL/WP composites, while reducing the mechanical strength. Moreover, experiments were performed to confirm the biocompatibility and osteogenic differentiation of human mesenchymal stem cells (MSCs) according to the component ratio of the scaffold. The results confirmed that the content of WP affects proliferation and osteogenic differentiation of MSCs. Based on the last day of the experiment, ie, the 14th day, the proliferation based on the amount of DNA was the best in the WP30% group, but all of the markers measured by PCR were the most expressed in the WP15% group. Conclusion These results suggest that the m_MS/PCL/WP composite is a promising candidate for use as a scaffold in cell-based bone regeneration. PMID:29520139

DNA mediated wire-like clusters of self-assembled TiO2 nanomaterials: supercapacitor and dye sensitized solar cell applications

NASA Astrophysics Data System (ADS)

Nithiyanantham, U.; Ramadoss, Ananthakumar; Ede, Sivasankara Rao; Kundu, Subrata

2014-06-01

A new route for the formation of wire-like clusters of TiO2 nanomaterials self-assembled in DNA scaffold within an hour of reaction time is reported. TiO2 nanomaterials are synthesized by the reaction of titanium-isopropoxide with ethanol and water in the presence of DNA under continuous stirring and heating at 60 °C. The individual size of the TiO2 NPs self-assembled in DNA and the diameter of the wires can be tuned by controlling the DNA to Ti-salt molar ratios and other reaction parameters. The eventual diameter of the individual particles varies between 15 +/- 5 nm ranges, whereas the length of the nanowires varies in the 2-3 μm range. The synthesized wire-like DNA-TiO2 nanomaterials are excellent materials for electrochemical supercapacitor and DSSC applications. From the electrochemical supercapacitor experiment, it was found that the TiO2 nanomaterials showed different specific capacitance (Cs) values for the various nanowires, and the order of Cs values are as follows: wire-like clusters (small size) > wire-like clusters (large size). The highest Cs of 2.69 F g-1 was observed for TiO2 having wire-like structure with small sizes. The study of the long term cycling stability of wire-like clusters (small size) electrode were shown to be stable, retaining ca. 80% of the initial specific capacitance, even after 5000 cycles. The potentiality of the DNA-TiO2 nanomaterials was also tested in photo-voltaic applications and the observed efficiency was found higher in the case of wire-like TiO2 nanostructures with larger sizes compared to smaller sizes. In future, the described method can be extended for the synthesis of other oxide based materials on DNA scaffold and can be further used in other applications like sensors, Li-ion battery materials or treatment for environmental waste water.A new route for the formation of wire-like clusters of TiO2 nanomaterials self-assembled in DNA scaffold within an hour of reaction time is reported. TiO2 nanomaterials are synthesized by the reaction of titanium-isopropoxide with ethanol and water in the presence of DNA under continuous stirring and heating at 60 °C. The individual size of the TiO2 NPs self-assembled in DNA and the diameter of the wires can be tuned by controlling the DNA to Ti-salt molar ratios and other reaction parameters. The eventual diameter of the individual particles varies between 15 +/- 5 nm ranges, whereas the length of the nanowires varies in the 2-3 μm range. The synthesized wire-like DNA-TiO2 nanomaterials are excellent materials for electrochemical supercapacitor and DSSC applications. From the electrochemical supercapacitor experiment, it was found that the TiO2 nanomaterials showed different specific capacitance (Cs) values for the various nanowires, and the order of Cs values are as follows: wire-like clusters (small size) > wire-like clusters (large size). The highest Cs of 2.69 F g-1 was observed for TiO2 having wire-like structure with small sizes. The study of the long term cycling stability of wire-like clusters (small size) electrode were shown to be stable, retaining ca. 80% of the initial specific capacitance, even after 5000 cycles. The potentiality of the DNA-TiO2 nanomaterials was also tested in photo-voltaic applications and the observed efficiency was found higher in the case of wire-like TiO2 nanostructures with larger sizes compared to smaller sizes. In future, the described method can be extended for the synthesis of other oxide based materials on DNA scaffold and can be further used in other applications like sensors, Li-ion battery materials or treatment for environmental waste water. Electronic supplementary information (ESI) available: The details about the instrument used for various characterizations and figures related to FE-SEM analysis, EDS analysis, photoluminescence (PL) and LASER Raman study are provided. Table related to FT-IR analysis is also provided. See DOI: 10.1039/c4nr01836b

Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing.

PubMed

Kshersagar, Jeevitaa; Kshirsagar, Ravi; Desai, Shashikant; Bohara, Raghvendra; Joshi, Meghnad

2018-03-05

Direct application of amnion has greater risk of immunological rejection and infection. Decellularization is an effective method to lower the risk of immune complications and infections. The bioreactor assembly with multiple cassettes was designed for decellurization of multiple amnions with different cell types simultaneously in single run. A detergent-based protocol was modified to remove all cellular components from amnion and diminish the DNA content to render it non-immunogenic. Amnion (n = 10) were treated with 2% sodium dodecyl sulphate (SDS), 5% dimethyl sulfoxide (DMSO) and 2% sodium deoxycholeate (SD). Decellularized amnion samples were analyzed by haematoxylin-eosin staining (HE), Alcian blue pH 1 (AB-pH-1), 4,6-diamnionidino-2-phenylindol (DAPI), Massion's trichrome stain, DNA quantification, mechanical testing and scanning electron microscopy (SEM). Histological analysis showed complete removal of cellular components and the histoarchitecture of scaffold remained intact. Amnion scaffold activated with platelet rich plasma (PRP) and calcium chloride composition supported better adherence to the wound than amnion alone. Only single application showed good healing. In vivo assessment of activated amnion revealed stable dressing. It has good promising outcome. At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse (Mus musculus). Cryopreservation had no adverse effect on the mechanical properties of the amnion scaffold. Cryopreservation of decellularized amnion by Dulbecco's modified eagle medium (DMEM) was expected to prepare off-the-shelf skin substitutes and preserve them to be immediately available upon request of patients' needs.

Three-Dimensional Coculture of Meniscal Cells and Mesenchymal Stem Cells in Collagen Type I Hydrogel on a Small Intestinal Matrix-A Pilot Study Toward Equine Meniscus Tissue Engineering.

PubMed

Kremer, Antje; Ribitsch, Iris; Reboredo, Jenny; Dürr, Julia; Egerbacher, Monika; Jenner, Florien; Walles, Heike

2017-05-01

Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for their qualification to be applied for meniscus tissue engineering. To investigate cell attachment of primary MC and MSC on SIS-muc matrix SEM pictures were performed. For molecular analysis, lyophilized samples of coculture constructs with different cell ratios (100% MC, 100% MSC, and 50% MC and 50% MSC, 20% MC, and 80% MSC) were digested and analyzed for DNA and GAG content. Active matrix remodeling of 3D coculture models was indicated by matrix metalloproteinases detection. For comparison of tissue-engineered constructs with the histologic architecture of natural equine menisci, paired lateral and medial menisci of 15 horses representing different age groups were examined. A meniscus phenotype with promising similarity to native meniscus tissue in its GAG/DNA expression in addition to Col I, Col II, and Aggrecan production was achieved using a scaffold composed of Col I gel on SIS-muc combined with a coculture of MC and MSC. The results encourage further development of this scaffold-cell combination for meniscus tissue engineering.

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

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

Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin

2010-02-12

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold,more » in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.« less

An additive manufacturing-based PCL-alginate-chondrocyte bioprinted scaffold for cartilage tissue engineering.

PubMed

Kundu, Joydip; Shim, Jin-Hyung; Jang, Jinah; Kim, Sung-Won; Cho, Dong-Woo

2015-11-01

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self-assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three-dimensional (3D) cell-printed scaffolds using layer-by-layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell-encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell-based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL-alginate gel constructs. PCL-alginate gels containing transforming growth factor-β (TGFβ) showed higher ECM formation. The 3D cell-printed scaffolds of PCL-alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL-alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell-printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.

Development of a carbazole-based fluorescence probe for G-quadruplex DNA: The importance of side-group effect on binding specificity

NASA Astrophysics Data System (ADS)

Wang, Ming-Qi; Ren, Gui-Ying; Zhao, Shuang; Lian, Guang-Chang; Chen, Ting-Ting; Ci, Yang; Li, Hong-Yao

2018-06-01

G-quadruplex DNAs are highly prevalent in the human genome and involved in many important biological processes. However, many aspects of their biological mechanism and significance still need to be elucidated. Therefore, the development of fluorescent probes for G-quadruplex detection is important for the basic research. We report here on the development of small molecular dyes designed on the basis of carbazole scaffold by introducing styrene-like substituents at its 9-position, for the purpose of G-quadruplex recognition. Results revealed that the side group on the carbazole scaffold was very important for their ability to selectively recognize G-quadruplex DNA structures. 1a with the pyridine side group displayed excellent fluorescence signal turn-on property for the specific discrimination of G-quadruplex DNAs against other nucleic acids. The characteristics of 1a were further investigated with UV-vis spectrophotometry, fluorescence, circular dichroism, FID assay and molecular docking to validate the selectivity, sensitivity and detailed binding mode toward G-quadruplex DNAs.

Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

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

Bucks, Michelle A.; O'Regan, Kevin J.; Murphy, Michael A.

2007-05-10

The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associatedmore » with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.« less

Novel Imidazoline Antimicrobial Scaffold That Inhibits DNA Replication with Activity against Mycobacteria and Drug Resistant Gram-Positive Cocci

PubMed Central

2015-01-01

Bacterial antimicrobial resistance is an escalating public health threat, yet the current antimicrobial pipeline remains alarmingly depleted, making the development of new antimicrobials an urgent need. Here, we identify a novel, potent, imidazoline antimicrobial compound, SKI-356313, with bactericidal activity against Mycobacterium tuberculosis and Gram-positive cocci, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). SKI-356313 is active in murine models of Streptococcus pneumoniae and MRSA infection and is potently bactericidal for both replicating and nonreplicating M. tuberculosis. Using a combination of genetics, whole genome sequencing, and a novel target ID approach using real time imaging of core macromolecular biosynthesis, we show that SKI-356313 inhibits DNA replication and displaces the replisome from the bacterial nucleoid. These results identify a new antimicrobial scaffold with a novel mechanism of action and potential therapeutic utility against nonreplicating M. tuberculosis and antibiotic resistant Gram-positive cocci. PMID:25222597

A Drug-Induced Hybrid Electrospun Poly-Capro-Lactone: Cell-Derived Extracellular Matrix Scaffold for Liver Tissue Engineering.

PubMed

Grant, Rhiannon; Hay, David C; Callanan, Anthony

2017-07-01

Liver transplant is the only treatment option for patients with end-stage liver failure, however, there are too few donor livers available for transplant. Whole organ tissue engineering presents a potential solution to the problem of rapidly escalating donor liver shortages worldwide. A major challenge for liver tissue engineers is the creation of a hepatocyte microenvironment; a niche in which liver cells can survive and function optimally. While polymers and decellularized tissues pose an attractive option for scaffold manufacturing, neither alone has thus far proved sufficient. This study exploited cell's native extracellular matrix (ECM) producing capabilities using two different histone deacetylase inhibitors, and combined these with the customizability and reproducibility of electrospun polymer scaffolds to produce a "best of both worlds" niche microenvironment for hepatocytes. The resulting hybrid poly-capro-lactone (PCL)-ECM scaffolds were validated using HepG2 hepatocytes. The hybrid PCL-ECM scaffolds maintained hepatocyte growth and function, as evidenced by metabolic activity and DNA quantitation. Mechanical testing revealed little significant difference between scaffolds, indicating that cells were responding to a biochemical and topographical profile rather than mechanical changes. Immunohistochemistry showed that the biochemical profile of the drug-derived and nondrug-derived ECMs differed in ratio of Collagen I, Laminin, and Fibronectin. Furthermore, the hybrid PCL-ECM scaffolds influence the gene expression profile of the HepG2s drastically; with expression of Albumin, Cytochrome P450 Family 1 Subfamily A Polypeptide 1, Cytochrome P450 Family 1 Subfamily A Polypeptide 2, Cytochrome P450 Family 3 Subfamily A Polypeptide 4, Fibronectin, Collagen I, and Collagen IV undergoing significant changes. Our results demonstrate that drug-induced hybrid PCL-ECM scaffolds provide a viable, translatable platform for creating a niche microenvironment for hepatocytes, supporting in vivo phenotype and function. These scaffolds offer great potential for tissue engineering and regenerative medicine strategies for whole organ tissue engineering.

Uncovering the self-assembly of DNA nanostructures by thermodynamics and kinetics.

PubMed

Wei, Xixi; Nangreave, Jeanette; Liu, Yan

2014-06-17

CONSPECTUS: DNA nanotechnology is one of the most flourishing interdisciplinary research fields. DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales. They can be used as scaffolds for organizing other nanoparticles, proteins, and chemical groups, leveraging their functions for creating complex bioinspired materials that may serve as smart drug delivery systems, in vitro or in vivo biomolecular computing platforms, and diagnostic devices. Achieving optimal structural features, efficient assembly protocols, and precise functional group positioning and modification requires a thorough understanding of the thermodynamics and kinetics of the DNA nanostructure self-assembly process. The most common real-time measurement strategies include monitoring changes in UV absorbance based on the hyperchromic effect of DNA, and the emission signal changes of DNA intercalating dyes or covalently conjugated fluorescent dyes/pairs that accompany temperature dependent structural changes. Thermodynamic studies of a variety of DNA nanostructures have been performed, from simple double stranded DNA formation to more complex origami assembly. The key parameters that have been evaluated in terms of stability and cooperativity include the overall dimensions, the folding path of the scaffold, crossover and nick point arrangement, length and sequence of single strands, and salt and ion concentrations. DNA tile-tile interactions through sticky end hybridization have also been analyzed, and the steric inhibition and rigidity of tiles turn out to be important factors. Many kinetic studies have also been reported, and most are based on double stranded DNA formation. A two-state assumption and the hypothesis of several intermediate states have been applied to determine the rate constant and activation energy of the DNA hybridization process. A few simulated models were proposed to represent the structural, mechanical, and kinetic properties of DNA hybridization. The kinetics of strand displacement reactions has also been studied as a special case of DNA hybridization. The thermodynamic and kinetic characteristics of DNA nanostructures have been exploited to develop rapid and isothermal annealing protocols. It is conceivable that a more thorough understanding of the DNA assembly process could be used to guide the structural design process and optimize the conditions for assembly, manipulation, and functionalization, thus benefiting both upstream design and downstream applications.

Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

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

Yang Yongmin; IgE Therapeutics, Inc., San Diego, CA 92121-2233; Barankiewicz, Teresa J.

2007-07-27

Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes onmore » either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.« less

DNA mediated wire-like clusters of self-assembled TiO₂ nanomaterials: supercapacitor and dye sensitized solar cell applications.

PubMed

Nithiyanantham, U; Ramadoss, Ananthakumar; Ede, Sivasankara Rao; Kundu, Subrata

2014-07-21

A new route for the formation of wire-like clusters of TiO₂ nanomaterials self-assembled in DNA scaffold within an hour of reaction time is reported. TiO₂ nanomaterials are synthesized by the reaction of titanium-isopropoxide with ethanol and water in the presence of DNA under continuous stirring and heating at 60 °C. The individual size of the TiO₂ NPs self-assembled in DNA and the diameter of the wires can be tuned by controlling the DNA to Ti-salt molar ratios and other reaction parameters. The eventual diameter of the individual particles varies between 15 ± 5 nm ranges, whereas the length of the nanowires varies in the 2-3 μm range. The synthesized wire-like DNA-TiO₂ nanomaterials are excellent materials for electrochemical supercapacitor and DSSC applications. From the electrochemical supercapacitor experiment, it was found that the TiO₂ nanomaterials showed different specific capacitance (Cs) values for the various nanowires, and the order of Cs values are as follows: wire-like clusters (small size) > wire-like clusters (large size). The highest Cs of 2.69 F g(-1) was observed for TiO₂ having wire-like structure with small sizes. The study of the long term cycling stability of wire-like clusters (small size) electrode were shown to be stable, retaining ca. 80% of the initial specific capacitance, even after 5000 cycles. The potentiality of the DNA-TiO₂ nanomaterials was also tested in photo-voltaic applications and the observed efficiency was found higher in the case of wire-like TiO₂ nanostructures with larger sizes compared to smaller sizes. In future, the described method can be extended for the synthesis of other oxide based materials on DNA scaffold and can be further used in other applications like sensors, Li-ion battery materials or treatment for environmental waste water.

New modulated design and synthesis of quercetin-Cu(II)/Zn(II)-Sn2(IV) scaffold as anticancer agents: in vitro DNA binding profile, DNA cleavage pathway and Topo-I activity.

PubMed

Tabassum, Sartaj; Zaki, Mehvash; Afzal, Mohd; Arjmand, Farukh

2013-07-21

New molecular topologies quercetin-Cu(II)-Sn2(IV) and Zn(II)-Sn2(IV)1 and 2 were designed and synthesized to act as potential cancer chemotherapeutic agents. Their interaction with CT DNA by UV-vis and fluorescence spectroscopy was evaluated revealing an electrostatic mode of binding. Quercetin complexes are capable of promoting DNA cleavage involving both single and double strand breaks. Complex 1 cleaved pBR322 DNA via an oxidative mechanism while 2 followed a hydrolytic pathway, accessible to the minor groove of the DNA double helix in accordance with molecular docking studies with the DNA duplex of sequence d(CGCGAATTCGCG)2 dodecamer demonstrating that the complex was stabilized by additional electrostatic and hydrogen bonding interactions with the DNA. ROS such as OH˙, H2O2 and O2˙(-) are the major metabolites responsible for chronic diseases such as cancer, respiratory disorders, HIV, and diabetes etc., therefore eliminating ROS by molecular scaffolds involving SOD enzymatic activity has emerged as a potential way to develop a novel class of drugs. Therefore, in vitro superoxide dismutase activity of redox active complex 1 was evaluated by using a xanthine/xanthine oxidase-NBT assay which showed an IC50 value of 2.26 μM. Moreover, the cytotoxicity of both the complexes were screened on a panel of human carcinoma cell lines (GI50 values <8.7 μM) which revealed that 1 has a better prospect of acting as a cancer chemotherapeutic agent, and to elucidate the mechanism of tumor inhibition, Topo-I enzymatic activity was carried out. Furthermore, molecular modeling studies were carried out to understand molecular features important for drug-enzyme interactions which offer new insights into the experimental model observations.

DNA Nanostructures as Models for Evaluating the Role of Enthalpy and Entropy in Polyvalent Binding

PubMed Central

Nangreave, Jeanette; Yan, Hao; Liu, Yan

2011-01-01

DNA nanotechnology allows the design and construction of nano-scale objects that have finely tuned dimensions, orientation, and structure with remarkable ease and convenience. Synthetic DNA nanostructures can be precisely engineered to model a variety of molecules and systems, providing the opportunity to probe very subtle biophysical phenomena. In this study, several such synthetic DNA nanostructures were designed to serve as models to study the binding behavior of polyvalent molecules and gain insight into how small changes to the ligand/receptor scaffolds, intended to vary their conformational flexibility, will affect their association equilibrium. This approach has yielded a quantitative identification of the roles of enthalpy and entropy in the affinity of polyvalent DNA nanostructure interactions, which exhibit an intriguing compensating effect. PMID:21381740

Engineering an Antibiotic to Fight Cancer: Optimization of the Novobiocin Scaffold to Produce Anti-Proliferative Agents

PubMed Central

Zhao, Huiping; Donnelly, Alison C.; Kusuma, Bhaskar R.; Brandt, Gary E. L.; Brown, Douglas; Rajewski, Roger A.; Vielhauer, George; Holzbeierlein, Jeffrey; Cohen, Mark S.; Blagg, Brian S. J.

2011-01-01

Development of the DNA gyrase inhibitor, novobiocin, into a selective Hsp90 inhibitor was accomplished through structural modifications to the amide side chain, coumarin ring, and sugar moiety. These species exhibit ~700-fold improved anti-proliferative activity versus the natural product as evaluated by cellular efficacies against breast, colon, prostate, lung, and other cancer cell lines. Utilization of structure–activity relationships established for three novobiocin synthons produced optimized scaffolds, which manifest mid-nanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition. PMID:21553822

Genome Wide Characterization of Simple Sequence Repeats in Cucumber

USDA-ARS?s Scientific Manuscript database

The whole genome sequence of the cucumber cultivar Gy14 was recently sequenced at 15× coverage with the Roche 454 Titanium technology. The microsatellite DNA sequences (simple sequence repeats, SSRs) in the assembled scaffolds were computationally explored and characterized. A total of 112,073 SSRs ...

Quantum dots–DNA bioconjugates: synthesis to applications

PubMed Central

2016-01-01

Semiconductor nanoparticles particularly quantum dots (QDs) are interesting alternatives to organic fluorophores for a range of applications such as biosensing, imaging and therapeutics. Addition of a programmable scaffold such as DNA to QDs further expands the scope and applicability of these hybrid nanomaterials in biology. In this review, the most important stages of preparation of QD–DNA conjugates for specific applications in biology are discussed. Special emphasis is laid on (i) the most successful strategies to disperse QDs in aqueous media, (ii) the range of different conjugation with detailed discussion about specific merits and demerits in each case, and (iii) typical applications of these conjugates in the context of biology. PMID:27920898

Influence of internal pore architecture on biological and mechanical properties of three-dimensional fiber deposited scaffolds for bone regeneration.

PubMed

Ostrowska, Barbara; Di Luca, Andrea; Szlazak, Karol; Moroni, Lorenzo; Swieszkowski, Wojciech

2016-04-01

Fused deposition modeling has been used to fabricate three-dimensional (3D) scaffolds for tissue engineering applications, because it allows to tailor their pore network. Despite the proven flexibility in doing so, a limited amount of studies have been performed to evaluate whether specific pore shapes have an influence on cell activity and tissue formation. Our study aimed at investigating the influence of internal pore architecture on the biological and mechanical properties of 3D scaffolds seeded with mesenchymal stromal cells. Polycaprolactone scaffolds with six different geometries were fabricated. The 3D samples were manufactured with different lay-down pattern of the fibers by varying the layer deposition angle from 0°/15°/30°, to 0°/30°/60°, 0°/45°/90°, 0°/60°/120°, 0°/75°/150°, and 0°/90°/180°. The scaffolds were investigated by scanning electron microscopy and micro computed tomographical analysis and displayed a fully interconnected pore network. Cell proliferation and differentiation toward the osteogenic lineage were evaluated by DNA, alkaline phosphatase activity, and polymerase chain reaction. The obtained scaffolds had structures with open porosity (50%-60%) and interconnected pores ranging from 380 to 400 µm. Changing the angle deposition affected significantly the mechanical properties of the scaffolds. With increasing the angle deposition between successive layers, the elastic modulus increased as well. Cellular studies also showed influence of the internal architecture on cell adhesion and proliferation within the 3D construct, yet limited influence on cell differentiation was observed. © 2016 Wiley Periodicals, Inc.

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

PubMed Central

Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

2016-01-01

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

Programming Self-Assembly of DNA Origami Honeycomb Two-Dimensional Lattices and Plasmonic Metamaterials.

PubMed

Wang, Pengfei; Gaitanaros, Stavros; Lee, Seungwoo; Bathe, Mark; Shih, William M; Ke, Yonggang

2016-06-22

Scaffolded DNA origami has proven to be a versatile method for generating functional nanostructures with prescribed sub-100 nm shapes. Programming DNA-origami tiles to form large-scale 2D lattices that span hundreds of nanometers to the micrometer scale could provide an enabling platform for diverse applications ranging from metamaterials to surface-based biophysical assays. Toward this end, here we design a family of hexagonal DNA-origami tiles using computer-aided design and demonstrate successful self-assembly of micrometer-scale 2D honeycomb lattices and tubes by controlling their geometric and mechanical properties including their interconnecting strands. Our results offer insight into programmed self-assembly of low-defect supra-molecular DNA-origami 2D lattices and tubes. In addition, we demonstrate that these DNA-origami hexagon tiles and honeycomb lattices are versatile platforms for assembling optical metamaterials via programmable spatial arrangement of gold nanoparticles (AuNPs) into cluster and superlattice geometries.

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair.

PubMed

Sterpone, Silvia; Cozzi, Renata

2010-07-25

It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer.

The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining.

PubMed

Lee, Younghyun; Li, Huizi Keiko; Masaoka, Aya; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A; Okayasu, Ryuichi

2016-10-01

PU-H71 is a purine-scaffold Hsp90 inhibitor developed to overcome limitations of conventional Hsp90 inhibitors. This study was designed to investigate the combined effect of PU-H71 and heavy ion irradiation on human tumor and normal cells. The effects of PU-H71 were determined by monitoring cell survival by colony formation, and DNA double-strand break (DSB) repair by γ-H2AX foci and immuno-blotting DSB repair proteins. The mode of cell death was evaluated by sub-G1 DNA content (as an indicator for apoptosis), and mitotic catastrophe. PU-H71 enhanced heavy ion irradiation-induced cell death in three human cancer cell lines, but the drug did not radiosensitize normal human fibroblasts. In irradiated tumor cells, PU-H71 increased the persistence of γ-H2AX foci, and it reduced RAD51 foci and phosphorylated DNA-PKcs, key DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ). In some tumor cell lines, PU-H71 altered the sub-G1 cell fraction and mitotic catastrophe following carbon ion irradiation. Our results demonstrate that PU-H71 sensitizes human cancer cells to heavy ion irradiation by inhibiting both HR and NHEJ DSB repair pathways. PU-H71 holds promise as a radiosensitizer for enhancing the efficacy of heavy ion radiotherapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DNA nanostructure-directed assembly of metal nanoparticle superlattices

NASA Astrophysics Data System (ADS)

Julin, Sofia; Nummelin, Sami; Kostiainen, Mauri A.; Linko, Veikko

2018-05-01

Structural DNA nanotechnology provides unique, well-controlled, versatile, and highly addressable motifs and templates for assembling materials at the nanoscale. These methods to build from the bottom-up using DNA as a construction material are based on programmable and fully predictable Watson-Crick base pairing. Researchers have adopted these techniques to an increasing extent for creating numerous DNA nanostructures for a variety of uses ranging from nanoelectronics to drug-delivery applications. Recently, an increasing effort has been put into attaching nanoparticles (the size range of 1-20 nm) to the accurate DNA motifs and into creating metallic nanostructures (typically 20-100 nm) using designer DNA nanoshapes as molds or stencils. By combining nanoparticles with the superior addressability of DNA-based scaffolds, it is possible to form well-ordered materials with intriguing and completely new optical, plasmonic, electronic, and magnetic properties. This focused review discusses the DNA structure-directed nanoparticle assemblies covering the wide range of different one-, two-, and three-dimensional systems.

Computer-aided design of DNA origami structures.

PubMed

Selnihhin, Denis; Andersen, Ebbe Sloth

2015-01-01

The DNA origami method enables the creation of complex nanoscale objects that can be used to organize molecular components and to function as reconfigurable mechanical devices. Of relevance to synthetic biology, DNA origami structures can be delivered to cells where they can perform complicated sense-and-act tasks, and can be used as scaffolds to organize enzymes for enhanced synthesis. The design of DNA origami structures is a complicated matter and is most efficiently done using dedicated software packages. This chapter describes a procedure for designing DNA origami structures using a combination of state-of-the-art software tools. First, we introduce the basic method for calculating crossover positions between DNA helices and the standard crossover patterns for flat, square, and honeycomb DNA origami lattices. Second, we provide a step-by-step tutorial for the design of a simple DNA origami biosensor device, from schematic idea to blueprint creation and to 3D modeling and animation, and explain how careful modeling can facilitate later experimentation in the laboratory.

Toward quantitative fluorescence microscopy with DNA origami nanorulers.

PubMed

Beater, Susanne; Raab, Mario; Tinnefeld, Philip

2014-01-01

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution. © 2014 Elsevier Inc. All rights reserved.

Examining multi-component DNA-templated nanostructures as imaging agents

NASA Astrophysics Data System (ADS)

Jaganathan, Hamsa

2011-12-01

Magnetic resonance imaging (MRI) is the leading non-invasive tool for disease imaging and diagnosis. Although MRI exhibits high spatial resolution for anatomical features, the contrast resolution is low. Imaging agents serve as an aid to distinguish different types of tissues within images. Gadolinium chelates, which are considered first generation designs, can be toxic to health, while ultra-small, superparamagnetic nanoparticles (NPs) have low tissue-targeting efficiency and rapid bio-distribution, resulting to an inadequate detection of the MRI signal and enhancement of image contrast. In order to improve the utility of MRI agents, the challenge in composition and structure needs to be addressed. One-dimensional (1D), superparamagnetic nanostructures have been reported to enhance magnetic and in vivo properties and therefore has a potential to improve contrast enhancement in MRI images. In this dissertation, the structure of 1D, multi-component NP chains, scaffolded on DNA, were pre-clinically examined as potential MRI agents. First, research was focused on characterizing and understanding the mechanism of proton relaxation for DNA-templated NP chains using nuclear magnetic resonance (NMR) spectrometry. Proton relaxation and transverse relaxivity were higher in multi-component NP chains compared to disperse NPs, indicating the arrangement of NPs on a 1D structure improved proton relaxation sensitivity. Second, in vitro evaluation for potential issues in toxicity and contrast efficiency in tissue environments using a 3 Tesla clinical MRI scanner was performed. Cell uptake of DNA-templated NP chains was enhanced after encapsulating the nanostructure with layers of polyelectrolytes and targeting ligands. Compared to dispersed NPs, DNA-templated NP chains improved MRI contrast in both the epithelial basement membrane and colon cancer tumors scaffolds. The last part of the project was focused on developing a novel MRI agent that detects changes in DNA methylation levels. The findings from this dissertation suggest that the structural arrangement of NPs on DNA significantly influenced their function and utility as MRI agents.

An electrospun scaffold integrating nucleic acid delivery for treatment of full thickness wounds

PubMed Central

Kobsa, Serge; Kristofik, Nina J.; Sawyer, Andrew J.; Bothwell, Alfred L.M.; Kyriakides, Themis R.; Saltzman, W. Mark

2013-01-01

We developed a multi-functional construct capable of controlled delivery of bioactive substances that can improve wound repair by supporting the intrinsic ability of the skin to heal. We synthesized electrospun scaffolds—composed of a blend of the degradable polymers poly(L-lactide) (PLA) or polycaprolactone (PCL)—that produce highly efficient non-viral in vivo gene delivery to cells in the wound bed, provide a protective barrier during early wound healing, and support cell migration and growth. This multi-functional material was tested for its influence on wound healing: scaffolds were loaded with plasmids encoding keratinocyte growth factor (KGF) and applied to full thickness wounds in mice. Compared to scaffolds with control plasmids, animals receiving the KGF plasmid-loaded scaffold produced significant enhancements in wound healing, which was quantified by improvements in the rate of wound re-epithelialization, keratinocyte proliferation, and granulation response. Further, we quantified the expression level of endogenous and plasmid-derived KGF in wound samples: qRT-PCR on wound sections revealed a correlation between the levels of plasmid-derived protein expression and histological analysis of wound healing, revealing an inverse relationship between the expression level of exogenous KGF and the size of the unhealed epithelial layer in wounds. Our findings suggest that engineered nanofiber PLA/PCL scaffolds are capable of highly efficient controlled DNA delivery and are promising materials for treatment of cutaneous wounds. PMID:23453058

In vitro culture of large bone substitutes in a new bioreactor: importance of the flow direction.

PubMed

Olivier, V; Hivart, Ph; Descamps, M; Hardouin, P

2007-09-01

New biomaterials combined with osteogenic cells are now being developed as an alternative to autogeneous bone grafts when the skeletal defect reaches a critical size. Yet, the size issue appears to be a key obstacle in the development of bone tissue engineering. Bioreactors are needed to allow the in vitro expansion of cells inside large bulk materials under appropriate conditions. However, no bioreactor has yet been designed for large-scale 3D structures and custom-made scaffolds. In this study, we evaluate the efficiency of a new bioreactor for the in vitro development of large bone substitutes, ensuring the perfusion of large ceramic scaffolds by the nutritive medium. The survival and proliferation of cells inside the scaffolds after 7 and 28 days in this dynamic culture system and the impact of the direction of the flow circulation are evaluated. The follow-up of glucose consumption, DNA quantification and microscopic evaluation all confirmed cell survival and proliferation for a sample under dynamic culture conditions, whereas static culture leads to the death of cells inside the scaffolds. Two directions of flow perfusion were assayed; the convergent direction leads to enhanced results compared to divergent flow.

Modeling of enhanced catalysis in multienzyme nanostructures: effect of molecular scaffolds, spatial organization, and concentration.

PubMed

Roberts, Christopher C; Chang, Chia-en A

2015-01-13

Colocalized multistep enzymatic reaction pathways within biological catabolic and metabolic processes occur with high yield and specificity. Spatial organization on membranes or surfaces may be associated with increased efficiency of intermediate substrate transfer. Using a new Brownian dynamics package, GeomBD, we explored the geometric features of a surface-anchored enzyme system by parallel coarse-grained Brownian dynamics simulations of substrate diffusion over microsecond (μs) to millisecond (ms) time scales. We focused on a recently developed glucose oxidase (GOx), horseradish peroxidase (HRP), and DNA origami-scaffold enzyme system, where the H2O2 substrate of HRP is produced by GOx. The results revealed and explained a significant advantage in catalytic enhancement by optimizing interenzyme distance and orientation in the presence of the scaffold model. The planar scaffold colocalized the enzymes and provided a diffusive barrier that enhanced substrate transfer probability, becoming more relevant with increasing interenzyme distance. The results highlight the importance of protein geometry in the proper assessment of distance and orientation dependence on the probability of substrate transfer. They shed light on strategies for engineering multienzyme complexes and further investigation of enhanced catalytic efficiency for substrate diffusion between membrane-anchoring proteins.

New branched DNA constructs.

PubMed

Chandra, Madhavaiah; Keller, Sascha; Gloeckner, Christian; Bornemann, Benjamin; Marx, Andreas

2007-01-01

The Watson-Crick base pairing of DNA is an advantageous phenomenon that can be exploited when using DNA as a scaffold for directed self-organization of nanometer-sized objects. Several reports have appeared in the literature that describe the generation of branched DNA (bDNA) with variable numbers of arms that self-assembles into predesigned architectures. These bDNA units are generated by using cleverly designed rigid crossover DNA molecules. Alternatively, bDNA can be generated by using synthetic branch points derived from either nucleoside or non-nucleoside building blocks. Branched DNA has scarcely been explored for use in nanotechnology or from self-assembling perspectives. Herein, we wish to report our results for the synthesis, characterization, and assembling properties of asymmetrical bDNA molecules that are able to generate linear and circular bDNA constructs. Our strategy for the generation of bDNA is based on a branching point that makes use of a novel protecting-group strategy. The bDNA units were generated by means of automated DNA synthesis methods and were used to generate novel objects by employing chemical and biological techniques. The entities generated might be useful building blocks for DNA-based nanobiotechnology.

Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells.

PubMed

Mitra, Debika; Whitehead, Jacklyn; Yasui, Osamu W; Leach, J Kent

2017-11-01

Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs in vitro to boost their bone forming capacity in vivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks in vivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days in vitro improves the quantity and quality of bone formation in vivo. This study highlights the need for optimizing in vitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of Adipose-Derived Stromal Cells and BMP12 on Intrasynovial Tendon Repair: A Biomechanical, Biochemical, and Proteomics Study

PubMed Central

Gelberman, Richard H.; Shen, Hua; Kormpakis, Ioannis; Rothrauff, Benjamin; Yang, Guang; Tuan, Rocky S.; Xia, Younan; Sakiyama-Elbert, Shelly; Silva, Matthew J.; Thomopoulos, Stavros

2016-01-01

The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor- and cell-based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin-based scaffold. Control groups consisted of repair-only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC+BMP12 treatment for range of motion or tensile properties outcomes versus repair-only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC+BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC+BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC+BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions. PMID:26445383

Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology

PubMed Central

Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

2012-01-01

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

Novel electrochemiluminescence of silver nanoclusters fabricated on triplex DNA scaffolds for label-free detection of biothiols.

PubMed

Feng, Lingyan; Wu, Li; Xing, Feifei; Hu, Lianzhe; Ren, Jinsong; Qu, Xiaogang

2017-12-15

Electrochemiluminescence (ECL) of metal nanoclusters and their application have been widely reported due to the good biocompatibility, fascinating electrocatalytic activity and so on. Using DNA as synthesis template opens new opportunities to modulate the physical properties of AgNCs. Triplex DNA has been reported for the site-specific, homogeneous and highly stable silver nanoclusters (AgNCs) fabrication from our recent research. Here we further explore their extraordinary ECL properties and applications in biosensor utilization. By reasonable design of DNA sequence, AgNCs were obtained in the predefined position of CG.C + sites of triplex DNA, and the ECL emission at a low potential was observed with this novel DNA template. Finally, a simple and label-free method was developed for biothiols detection based on the enhanced catalytic reaction and a robust interaction between the triplex-AgNCs and cysteine, by influencing the microenvironment provided by DNA template. Copyright © 2017 Elsevier B.V. All rights reserved.

Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography

DOE PAGES

Lei, Dongsheng; Marras, Alexander E.; Liu, Jianfang; ...

2018-02-09

Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. In this paper, we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~6-14 nm resolution. The statisticalmore » analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.« less

Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography

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

Lei, Dongsheng; Marras, Alexander E.; Liu, Jianfang

Scaffolded DNA origami has proven to be a powerful and efficient technique to fabricate functional nanomachines by programming the folding of a single-stranded DNA template strand into three-dimensional (3D) nanostructures, designed to be precisely motion-controlled. Although two-dimensional (2D) imaging of DNA nanomachines using transmission electron microscopy and atomic force microscopy suggested these nanomachines are dynamic in 3D, geometric analysis based on 2D imaging was insufficient to uncover the exact motion in 3D. In this paper, we use the individual-particle electron tomography method and reconstruct 129 density maps from 129 individual DNA origami Bennett linkage mechanisms at ~6-14 nm resolution. The statisticalmore » analyses of these conformations lead to understanding the 3D structural dynamics of Bennett linkage mechanisms. Moreover, our effort provides experimental verification of a theoretical kinematics model of DNA origami, which can be used as feedback to improve the design and control of motion via optimized DNA sequences and routing.« less

Comparison of 3D-Printed Poly-ɛ-Caprolactone Scaffolds Functionalized with Tricalcium Phosphate, Hydroxyapatite, Bio-Oss, or Decellularized Bone Matrix^.

PubMed

Nyberg, Ethan; Rindone, Alexandra; Dorafshar, Amir; Grayson, Warren L

2017-06-01

Three-dimensional (3D)-printing facilitates rapid, custom manufacturing of bone scaffolds with a wide range of material choices. Recent studies have demonstrated the potential for 3D-printing bioactive (i.e., osteo-inductive) scaffolds for use in bone regeneration applications. In this study, we 3D-printed porous poly-ɛ-caprolactone (PCL) scaffolds using a fused deposition modeling (FDM) process and functionalized them with mineral additives that have been widely used commercially and clinically: tricalcium phosphate (TCP), hydroxyapatite (HA), Bio-Oss (BO), or decellularized bone matrix (DCB). We assessed the "print quality" of the composite scaffolds and found that the print quality of PCL-TCP, PCL-BO, and PCL-DCB measured ∼0.7 and was statistically lower than PCL and PCL-HA scaffolds (∼0.8). We found that the incorporation of mineral particles did not significantly decrease the compressive modulus of the graft, which was on the order of 260 MPa for solid blocks and ranged from 32 to 83 MPa for porous scaffolds. Raman spectroscopy revealed the surfaces of the scaffolds maintained the chemical profile of their dopants following the printing process. We evaluated the osteo-inductive properties of each scaffold composite by culturing adipose-derived stromal/stem cells in vitro and assessing their differentiation into osteoblasts. The calcium content (normalized to DNA) increased significantly in PCL-TCP (p < 0.05), PCL-BO (p < 0.001), and PCL-DCB (p < 0.0001) groups relative to PCL only. The calcium content also increased in PCL-HA but was not statistically significant (p > 0.05). Collagen 1 expression was 10-fold greater than PCL in PCL-BO and PCL-DCB (p < 0.05) and osteocalcin expression was 10-fold greater in PCL-BO and PCL-DCB (p < 0.05) as measured by quantitative-real time-polymerase chain reaction. This study suggests that PCL-BO and PCL-DCB hybrid material may be advantageous for bone healing applications over PCL-HA or PCL-TCP blends.

Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

PubMed Central

Giordano, Roberto; Magnano, Anna Rosa; Zaccagnini, Germana; Pittoggi, Carmine; Moscufo, Nicola; Lorenzini, Rodolfo; Spadafora, Corrado

2000-01-01

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development. PMID:10725323

Treating Pompe Disease

ERIC Educational Resources Information Center

Bokor, Julie; Joseph, Drew; Darwiche, Houda

2015-01-01

One of the crosscutting concepts in science is cause and effect. A disease model can provide understanding of cause and effect, as teachers scaffold student thinking from molecular changes in the DNA to visible traits in the organism. The project described in this article uses Pompe disease, a rare recessive disorder, as a model of cause and…

Targeting MED1 LxxLL Motifs for Tissue-Selective Treatment of Human Breast Cancer

DTIC Science & Technology

2012-09-01

his colleagues have successfully conjugated malachite green (MG) aptamer to RNA nanoparticles characterized by a three-way junction (3WJ) pRNA motif...nanoparticle harboring malachite green (MG) aptamer, survivin siRNA and folate-DNA/RNA sequence for targeting delivery, using 3WJ-pRNA as scaffolds. Figure

Development of a carbazole-based fluorescence probe for G-quadruplex DNA: The importance of side-group effect on binding specificity.

PubMed

Wang, Ming-Qi; Ren, Gui-Ying; Zhao, Shuang; Lian, Guang-Chang; Chen, Ting-Ting; Ci, Yang; Li, Hong-Yao

2018-06-15

G-quadruplex DNAs are highly prevalent in the human genome and involved in many important biological processes. However, many aspects of their biological mechanism and significance still need to be elucidated. Therefore, the development of fluorescent probes for G-quadruplex detection is important for the basic research. We report here on the development of small molecular dyes designed on the basis of carbazole scaffold by introducing styrene-like substituents at its 9-position, for the purpose of G-quadruplex recognition. Results revealed that the side group on the carbazole scaffold was very important for their ability to selectively recognize G-quadruplex DNA structures. 1a with the pyridine side group displayed excellent fluorescence signal turn-on property for the specific discrimination of G-quadruplex DNAs against other nucleic acids. The characteristics of 1a were further investigated with UV-vis spectrophotometry, fluorescence, circular dichroism, FID assay and molecular docking to validate the selectivity, sensitivity and detailed binding mode toward G-quadruplex DNAs. Copyright © 2018 Elsevier B.V. All rights reserved.

Spatial organization of heterologous metabolic system in vivo based on TALE.

PubMed

Zhu, Lv-yun; Qiu, Xin-Yuan; Zhu, Ling-Yun; Wu, Xiao-Min; Zhang, Yuan; Zhu, Qian-Hui; Fan, Dong-Yu; Zhu, Chu-Shu; Zhang, Dong-Yi

2016-05-17

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

PubMed

Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

2014-11-26

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

Simple design for DNA nanotubes from a minimal set of unmodified strands: rapid, room-temperature assembly and readily tunable structure.

PubMed

Hamblin, Graham D; Hariri, Amani A; Carneiro, Karina M M; Lau, Kai L; Cosa, Gonzalo; Sleiman, Hanadi F

2013-04-23

DNA nanotubes have great potential as nanoscale scaffolds for the organization of materials and the templation of nanowires and as drug delivery vehicles. Current methods for making DNA nanotubes either rely on a tile-based step-growth polymerization mechanism or use a large number of component strands and long annealing times. Step-growth polymerization gives little control over length, is sensitive to stoichiometry, and is slow to generate long products. Here, we present a design strategy for DNA nanotubes that uses an alternative, more controlled growth mechanism, while using just five unmodified component strands and a long enzymatically produced backbone. These tubes form rapidly at room temperature and have numerous, orthogonal sites available for the programmable incorporation of arrays of cargo along their length. As a proof-of-concept, cyanine dyes were organized into two distinct patterns by inclusion into these DNA nanotubes.

Construction of a Holliday Junction in Small Circular DNA Molecules for Stable Motifs and Two-Dimensional Lattices.

PubMed

Guo, Xin; Wang, Xue-Mei; Wei, Shuai; Xiao, Shou-Jun

2018-04-12

Design rules for DNA nanotechnology have been mostly learnt from using linear single-stranded (ss) DNA as the source material. For example, the core structure of a typical DAO (double crossover, antiparallel, odd half-turns) tile for assembling 2D lattices is constructed from only two linear ss-oligonucleotide scaffold strands, similar to two ropes making a square knot. Herein, a new type of coupled DAO (cDAO) tile and 2D lattices of small circular ss-oligonucleotides as scaffold strands and linear ss-oligonucleotides as staple strands are reported. A cDAO tile of cDAO-c64nt (c64nt: circular 64 nucleotides), shaped as a solid parallelogram, is constructed with a Holliday junction (HJ) at the center and two HJs at both poles of a c64nt; similarly, cDAO-c84nt, shaped as a crossed quadrilateral composed of two congruent triangles, is formed with a HJ at the center and four three-way junctions at the corners of a c84nt. Perfect 2D lattices were assembled from cDAO tiles: infinite nanostructures of nanoribbons, nanotubes, and nanorings, and finite nanostructures. The structural relationship between the visible lattices imaged by AFM and the corresponding invisible secondary and tertiary molecular structures of HJs, inclination angle of hydrogen bonds against the double-helix axis, and the chirality of the tile can be interpreted very well. This work could shed new light on DNA nanotechnology with unique circular tiles. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photothermal stress triggered by near infrared-irradiated carbon nanotubes promotes bone deposition in rat calvarial defects.

PubMed

Yanagi, Tsukasa; Kajiya, Hiroshi; Kawaguchi, Minoru; Kido, Hirofumi; Fukushima, Tadao

2015-03-01

The bone regenerative healing process is often prolonged, with a high risk of infection particularly in elderly and diseased patients. A reduction in healing process time usually requires mechanical stress devices, chemical cues, or laser/thermal therapies. Although these approaches have been used extensively for the reduction of bone healing time, the exact mechanisms involved in thermal stress-induced bone regeneration remain unclear. In this study, we investigated the effect of optimal hyperthermia on rat calvarial defects in vivo and on osteogenesis in vitro. Photothermal stress stimulation was carried out using a new photothermal device, composed of an alginate gel including in carbon nanotubes and their irradiator with near-infrared light. Photothermal stress (15 min at 42℃, every day), trigged by near-infrared-induced carbon nanotube, promoted bone deposition in critical-sized calvarial defects compared with nonthermal stress controls. We recently reported that our novel DNA/protamine complex scaffold induces bone regeneration in calvarial defects. In this study, photothermal stress upregulated bone deposition in DNA/protamine-engrafted calvarial defects. Furthermore, photothermal stress significantly induced expression of osteogenic related genes in a time-dependent manner, including alkaline phosphatase, osterix, and osteocalcin. This was observed in DNA/protamine cells, which were expanded from regenerated tissue engrafted into the DNA/protamine scaffold, as well as in human MG63 preosteoblasts. In summary, this novel carbon nanotube-based photothermal stress approach upregulated expression of osteogenic-related genes in preosteoblasts, resulting in promotion of mineral deposition for enhanced bone repair. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Two-photon polymerization technique for microfabrication of CAD-designed 3D scaffolds from commercially available photosensitive materials.

PubMed

Ovsianikov, Aleksandr; Schlie, Sabrina; Ngezahayo, Anaclet; Haverich, Axel; Chichkov, Boris N

2007-01-01

We report on recent advances in the fabrication of three-dimensional (3D) scaffolds for tissue engineering and regenerative medicine constructs using a two-photon polymerization technique (2PP). 2PP is a novel CAD/CAM technology allowing the fabrication of any computer-designed 3D structure from a photosensitive polymeric material. The flexibility of this technology and the ability to precisely define 3D construct geometry allows issues associated with vascularization and patient-specific tissue fabrication to be directly addressed. The fabrication of reproducible scaffold structures by 2PP is important for systematic studies of cellular processes and better understanding of in vitro tissue formation. In this study, 2PP was applied for the generation of 3D scaffold-like structures, using the photosensitive organic-inorganic hybrid polymer ORMOCER (ORganically MOdified CERamics) and epoxy-based SU8 materials. By comparing the proliferation rates of cells grown on flat material surfaces and under control conditions, it was demonstrated that ORMOCER and SU8 are not cytotoxic. Additional tests show that the DNA strand breaking of GFSHR-17 granulosa cells was not affected by the presence of ORMOCER. Furthermore, gap junction conductance measurements revealed that ORMOCER did not alter the formation of cell-cell junctions, critical for functional tissue growth. The possibilities of seeding 3D structures with cells were analysed. These studies demonstrate the great potential of 2PP technique for the manufacturing of scaffolds with controlled topology and properties.

Matrix composition and mechanics of decellularized lung scaffolds.

PubMed

Petersen, Thomas H; Calle, Elizabeth A; Colehour, Maegen B; Niklason, Laura E

2012-01-01

The utility of decellularized native tissues for tissue engineering has been widely demonstrated. Here, we examine the production of decellularized lung scaffolds from native rodent lung using two different techniques, principally defined by use of either the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or sodium dodecyl sulfate (SDS). All viable cellular material is removed, including at least 99% of DNA. Histochemical staining and mechanical testing indicate that collagen and elastin are retained in the decellularized matrices with CHAPS-based decellularization, while SDS-based decellularization leads to loss of collagen and decline in mechanical strength. Quantitative assays confirm that most collagen is retained with CHAPS treatment but that about 80% of collagen is lost with SDS treatment. In contrast, for both detergent methods, at least 60% of elastin content is lost along with about 95% of native proteoglycan content. Mechanical testing of the decellularized scaffolds indicates that they are mechanically similar to native lung using CHAPS decellularization, including retained tensile strength and elastic behavior, demonstrating the importance of collagen and elastin in lung mechanics. With SDS decellularization, the mechanical integrity of scaffolds is significantly diminished with some loss of elastic function as well. Finally, a simple theoretical model of peripheral lung matrix mechanics is consonant with our experimental findings. This work demonstrates the feasibility of producing a decellularized lung scaffold that can be used to study lung matrix biology and mechanics, independent of the effects of cellular components. Copyright © 2011 S. Karger AG, Basel.

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

PubMed

Rath, Subha N; Strobel, Leonie A; Arkudas, Andreas; Beier, Justus P; Maier, Anne-Kathrin; Greil, Peter; Horch, Raymund E; Kneser, Ulrich

2012-10-01

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

PubMed Central

Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Dane

2016-01-01

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. PMID:27582495

Strand-invading linear probe combined with unmodified PNA.

PubMed

Asanuma, Hiroyuki; Niwa, Rie; Akahane, Mariko; Murayama, Keiji; Kashida, Hiromu; Kamiya, Yukiko

2016-09-15

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40°C at 100mM NaCl concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nucleic Acid Nanostructures: Bottom-Up Control of Geometry on the Nanoscale

PubMed Central

Seeman, Nadrian C.; Lukeman, Philip S.

2012-01-01

DNA may seem an unlikely molecule from which to build nanostructures, but this is not correct. The specificity of interaction that enables DNA to function so successfully as genetic material also enables its use as a smart molecule for construction on the nanoscale. The key to using DNA for this purpose is the design of stable branched molecules, which expand its ability to interact specifically with other nucleic acid molecules. The same interactions used by genetic engineers can be used to make cohesive interactions with other DNA molecules that lead to a variety of new species. Branched DNA molecules are easy to design, and the can assume a variety of structural motifs. These can be used for purposes both of specific construction, such as polyhedra, and for the assembly of topological targets. A variety of two-dimensional periodic arrays with specific patterns have been made. DNA nanomechanical devices have been built with a series of different triggers, small molecules, nucleic acid molecules and proteins. Recently, progress has been made in self-replication of DNA nano-constructs, and in the scaffolding of other species into DNA arrangements. PMID:25152542

Interlocked DNA nanostructures controlled by a reversible logic circuit.

PubMed

Li, Tao; Lohmann, Finn; Famulok, Michael

2014-09-17

DNA nanostructures constitute attractive devices for logic computing and nanomechanics. An emerging interest is to integrate these two fields and devise intelligent DNA nanorobots. Here we report a reversible logic circuit built on the programmable assembly of a double-stranded (ds) DNA [3]pseudocatenane that serves as a rigid scaffold to position two separate branched-out head-motifs, a bimolecular i-motif and a G-quadruplex. The G-quadruplex only forms when preceded by the assembly of the i-motif. The formation of the latter, in turn, requires acidic pH and unhindered mobility of the head-motif containing dsDNA nanorings with respect to the central ring to which they are interlocked, triggered by release oligodeoxynucleotides. We employ these features to convert the structural changes into Boolean operations with fluorescence labelling. The nanostructure behaves as a reversible logic circuit consisting of tandem YES and AND gates. Such reversible logic circuits integrated into functional nanodevices may guide future intelligent DNA nanorobots to manipulate cascade reactions in biological systems.

Interlocked DNA nanostructures controlled by a reversible logic circuit

PubMed Central

Li, Tao; Lohmann, Finn; Famulok, Michael

2014-01-01

DNA nanostructures constitute attractive devices for logic computing and nanomechanics. An emerging interest is to integrate these two fields and devise intelligent DNA nanorobots. Here we report a reversible logic circuit built on the programmable assembly of a double-stranded (ds) DNA [3]pseudocatenane that serves as a rigid scaffold to position two separate branched-out head-motifs, a bimolecular i-motif and a G-quadruplex. The G-quadruplex only forms when preceded by the assembly of the i-motif. The formation of the latter, in turn, requires acidic pH and unhindered mobility of the head-motif containing dsDNA nanorings with respect to the central ring to which they are interlocked, triggered by release oligodeoxynucleotides. We employ these features to convert the structural changes into Boolean operations with fluorescence labelling. The nanostructure behaves as a reversible logic circuit consisting of tandem YES and AND gates. Such reversible logic circuits integrated into functional nanodevices may guide future intelligent DNA nanorobots to manipulate cascade reactions in biological systems. PMID:25229207

Alkylpurine glycosylase D employs DNA sculpting as a strategy to extrude and excise damaged bases.

PubMed

Kossmann, Bradley; Ivanov, Ivaylo

2014-07-01

Alkylpurine glycosylase D (AlkD) exhibits a unique base excision strategy. Instead of interacting directly with the lesion, the enzyme engages the non-lesion DNA strand. AlkD induces flipping of the alkylated and opposing base accompanied by DNA stack compression. Since this strategy leaves the alkylated base solvent exposed, the means to achieve enzymatic cleavage had remained unclear. We determined a minimum energy path for flipping out a 3-methyl adenine by AlkD and computed a potential of mean force along this path to delineate the energetics of base extrusion. We show that AlkD acts as a scaffold to stabilize three distinct DNA conformations, including the final extruded state. These states are almost equivalent in free energy and separated by low barriers. Thus, AlkD acts by sculpting the global DNA conformation to achieve lesion expulsion from DNA. N-glycosidic bond scission is then facilitated by a backbone phosphate group proximal to the alkylated base.

ATP Depletion Blocks Herpes Simplex Virus DNA Packaging and Capsid Maturation

PubMed Central

Dasgupta, Anindya; Wilson, Duncan W.

1999-01-01

During herpes simplex virus (HSV) assembly, immature procapsids must expel their internal scaffold proteins, transform their outer shell to form mature polyhedrons, and become packaged with the viral double-stranded (ds) DNA genome. A large number of virally encoded proteins are required for successful completion of these events, but their molecular roles are poorly understood. By analogy with the dsDNA bacteriophage we reasoned that HSV DNA packaging might be an ATP-requiring process and tested this hypothesis by adding an ATP depletion cocktail to cells accumulating unpackaged procapsids due to the presence of a temperature-sensitive lesion in the HSV maturational protease UL26. Following return to permissive temperature, HSV capsids were found to be unable to package DNA, suggesting that this process is indeed ATP dependent. Surprisingly, however, the display of epitopes indicative of capsid maturation was also inhibited. We conclude that either formation of these epitopes directly requires ATP or capsid maturation is normally arrested by a proofreading mechanism until DNA packaging has been successfully completed. PMID:9971781

Programme DNA Lattices: Design, Synthesis and Applications

DTIC Science & Technology

2006-02-01

N.C. Seeman, Nanotecnologia basada en ADN, Revista de Química 19, 11- 20, (2004), in Spanish. [SLM+00] R. Sha, F. Liu, D.P. Millar N.C. Seeman...Scaffolding, NanoLetters 4, 2343-2347, (2004) 47. G. Serrano & N.C. Seeman, Nanotecnologia basada en ADN, Revista de Química 19, 11- 20, (2004), in

Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-05-01

DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14-18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment.

Tissue factor activity and ECM-related gene expression in human aortic endothelial cells grown on electrospun biohybrid scaffolds.

PubMed

Han, Jingjia; Gerstenhaber, Jonathan A; Lazarovici, Philip; Lelkes, Peter I

2013-05-13

All blood vessels are lined with a quiescent endothelium, which aids in regulating regular blood flow and avoiding thrombus formation. Current attempts at replacing diseased blood vessels frequently fail due to the intrinsic thrombogenicity of the materials used as vascular grafts. In extending our previous work where we introduced a new candidate scaffolds for vascular grafts electrospun from a blend solution of PLGA, gelatin, and elastin (PGE), this study aimed to evaluate the potential of PGE scaffolds to support nonthrombogenic monolayers of primary isolates of human aortic endothelial cells (HAECs), as assessed by a combination of biochemical, molecular, and bioinformatics-based analyses. After 24 h of culture on 3-D fibrous PGE scaffolds, HAECs formed a confluent, nonthrombogenic, and physiologically competent monolayer, as assessed by tissue factor (TF) gene expression and protein activity assays. The levels of TF mRNA/protein activity in HAECs grown on PGE scaffolds were similar to those on gelatin or collagen IV-coated 2-D surfaces. In addition, bioinformatics-based analysis of a focused microarray containing 84 ECM-related cDNA probes demonstrated that HAECs essentially expressed a histotypic ECM-related "transcriptome" on PGE scaffolds, where cells were more quiescent than cells cultured on 2-D coverslips coated with gelatin (a well-known "inert" substrate for conventional EC culture), but less so than on 2-D PGE films. These data suggest an important role for nanorough substrates (PGE films) in passivating endothelial cells and confirm the crucial effect of substrate composition in this process. Principal component analysis of microarray data on the above substrates (including collagen IV) implied that substrate composition plays a greater role than surface topography in affecting the endothelial ECM-related "transcriptome". Taken together, our findings suggest that electrospun PGE scaffolds are potentially suitable for application in small diameter vascular tissue engineering.

Enzyme-guided DNA Sewing Architecture

PubMed Central

Song, In Hyun; Shin, Seung Won; Park, Kyung Soo; Lansac, Yves; Jang, Yun Hee; Um, Soong Ho

2015-01-01

With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs have been used as a functional building block for novel nanoarchitecture. In particular, a self-assembly of long DNA molecules via a piece DNA staple has been utilized to attain such constructs. However, it needs many talented prerequisites (e.g., complicated computer program) with fewer yields of products. In addition, it has many limitations to overcome: for instance, (i) thermal instability under moderate environments and (ii) restraint in size caused by the restricted length of scaffold strands. Alternatively, the enzymatic sewing linkage of short DNA blocks is simply designed into long DNA assemblies but it is more error-prone due to the undeveloped sequence data. Here, we present, for the first time, a comprehensive study for directly combining DNA structures into higher DNA sewing constructs through the 5′-end cohesive ligation of T4 enzyme. Inspired by these achievements, the synthesized DNA nanomaterials were also utilized for effective detection and real-time diagnosis of cancer-specific and cytosolic RNA markers. This generalized protocol for generic DNA sewing is expected to be useful in several DNA nanotechnology as well as any nucleic acid-related fields. PMID:26634810

Enzyme-guided DNA Sewing Architecture

NASA Astrophysics Data System (ADS)

Song, In Hyun; Shin, Seung Won; Park, Kyung Soo; Lansac, Yves; Jang, Yun Hee; Um, Soong Ho

2015-12-01

With the advent of nanotechnology, a variety of nanoarchitectures with varied physicochemical properties have been designed. Owing to the unique characteristics, DNAs have been used as a functional building block for novel nanoarchitecture. In particular, a self-assembly of long DNA molecules via a piece DNA staple has been utilized to attain such constructs. However, it needs many talented prerequisites (e.g., complicated computer program) with fewer yields of products. In addition, it has many limitations to overcome: for instance, (i) thermal instability under moderate environments and (ii) restraint in size caused by the restricted length of scaffold strands. Alternatively, the enzymatic sewing linkage of short DNA blocks is simply designed into long DNA assemblies but it is more error-prone due to the undeveloped sequence data. Here, we present, for the first time, a comprehensive study for directly combining DNA structures into higher DNA sewing constructs through the 5‧-end cohesive ligation of T4 enzyme. Inspired by these achievements, the synthesized DNA nanomaterials were also utilized for effective detection and real-time diagnosis of cancer-specific and cytosolic RNA markers. This generalized protocol for generic DNA sewing is expected to be useful in several DNA nanotechnology as well as any nucleic acid-related fields.

Enzyme-crosslinked gene-activated matrix for the induction of mesenchymal stem cells in osteochondral tissue regeneration.

PubMed

Lee, Yi-Hsuan; Wu, Hsi-Chin; Yeh, Chia-Wei; Kuan, Chen-Hsiang; Liao, Han-Tsung; Hsu, Horng-Chaung; Tsai, Jui-Che; Sun, Jui-Sheng; Wang, Tzu-Wei

2017-11-01

The development of osteochondral tissue engineering is an important issue for the treatment of traumatic injury or aging associated joint disease. However, the different compositions and mechanical properties of cartilage and subchondral bone show the complexity of this tissue interface, making it challenging for the design and fabrication of osteochondral graft substitute. In this study, a bilayer scaffold is developed to promote the regeneration of osteochondral tissue within a single integrated construct. It has the capacity to serve as a gene delivery platform to promote transfection of human mesenchymal stem cells (hMSCs) and the functional osteochondral tissues formation. For the subchondral bone layer, the bone matrix with organic (type I collagen, Col) and inorganic (hydroxyapatite, Hap) composite scaffold has been developed through mineralization of hydroxyapatite nanocrystals oriented growth on collagen fibrils. We also prepare multi-shell nanoparticles in different layers with a calcium phosphate core and DNA/calcium phosphate shells conjugated with polyethyleneimine to act as non-viral vectors for delivery of plasmid DNA encoding BMP2 and TGF-β3, respectively. Microbial transglutaminase is used as a cross-linking agent to crosslink the bilayer scaffold. The ability of this scaffold to act as a gene-activated matrix is demonstrated with successful transfection efficiency. The results show that the sustained release of plasmids from gene-activated matrix can promote prolonged transgene expression and stimulate hMSCs differentiation into osteogenic and chondrogenic lineages by spatial and temporal control within the bilayer composite scaffold. This improved delivery method may enhance the functionalized composite graft to accelerate healing process for osteochondral tissue regeneration. In this study, a gene-activated matrix (GAM) to promote the growth of both cartilage and subchondral bone within a single integrated construct is developed. It has the capacity to promote transfection of human mesenchymal stem cells (hMSCs) and the functional osteochondral tissues formation. The results show that the sustained release of plasmids including TGF-beta and BMP-2 from GAM could promote prolonged transgene expression and stimulate hMSCs differentiation into the osteogenic and chondrogenic lineages by spatial control manner. This improved delivery method should enhance the functionalized composite graft to accelerate healing process in vitro and in vivo for osteochondral tissue regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Designing a Bio-responsive Robot from DNA Origami

PubMed Central

Ben-Ishay, Eldad; Abu-Horowitz, Almogit; Bachelet, Ido

2013-01-01

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect. Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (>7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 . The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient. In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings. PMID:23893007

Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice

PubMed Central

Ghosh, Somnath; Canugovi, Chandrika; Yoon, Jeong Seon; Wilson, David M.; Croteau, Deborah L.; Mattson, Mark P.; Bohr, Vilhelm A.

2017-01-01

Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our lab showed that mice lacking the BER glycosylases Ogg1 or Neil1 recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of one of the two alleles of Xrcc1, which encodes a non-enzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1+/− mice than in wild-type mice. Additionally, a greater percentage of Xrcc1+/− mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared to controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery. PMID:25971543

Optimization of alginate purification using polyvinylidene difluoride membrane filtration: Effects on immunogenicity and biocompatibility of three-dimensional alginate scaffolds

PubMed Central

Sondermeijer, Hugo P; Witkowski, Piotr; Woodland, David; Seki, Tetsunori; Aangenendt, Frank J; van der Laarse, Arnoud; Itescu, Silviu; Hardy, Mark A

2017-01-01

Sodium alginate is an effective biomaterial for tissue engineering applications. Non-purified alginate is contaminated with protein, lipopolysaccharide, DNA, and RNA, which could elicit adverse immunological reactions. We developed a purification protocol to generate biocompatible alginate based on (a) activated charcoal treatment, (b) use of hydrophobic membrane filtration (we used hydrophobic polyvinylidene difluoride membranes to remove organic contaminants), (c) dialysis, and finally (d) ethanol precipitation. Using this approach, we could omit pre-treatment with chloroform and significantly reduce the quantities of reagents used. Purification resulted in reduction of residual protein by 70% down to 0.315 mg/g, DNA by 62% down to 1.28 μg/g, and RNA by 61% down to less than 10 μg/g, respectively. Lipopolysaccharide levels were reduced by >90% to less than 125 EU/g. Purified alginate did not induce splenocyte proliferation in vitro. Three-dimensional scaffolds generated from purified alginate did not elicit a significant foreign body reaction, fibrotic overgrowth, or macrophage infiltration 4 weeks after implantation. This study describes a simplified and economical alginate purification method that results in alginate purity, which meets clinically useful criteria. PMID:27114440

Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source.

PubMed

Van Eijk, F; Saris, D B F; Riesle, J; Willems, W J; Van Blitterswijk, C A; Verbout, A J; Dhert, W J A

2004-01-01

Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p < 0.01) and the highest collagen production (p < 0.05 for the two highest seeding densities). Scaffolds seeded with ACL fibroblasts showed the lowest DNA content and collagen production. Accordingly, BMSCs appear to be the most suitable cells for further study and development of tissue-engineered ligament.

Discovery, SAR, and X-ray Binding Mode Study of BCATm Inhibitors from a Novel DNA-Encoded Library

PubMed Central

2015-01-01

As a potential target for obesity, human BCATm was screened against more than 14 billion DNA encoded compounds of distinct scaffolds followed by off-DNA synthesis and activity confirmation. As a consequence, several series of BCATm inhibitors were discovered. One representative compound (R)-3-((1-(5-bromothiophene-2-carbonyl)pyrrolidin-3-yl)oxy)-N-methyl-2′-(methylsulfonamido)-[1,1′-biphenyl]-4-carboxamide (15e) from a novel compound library synthesized via on-DNA Suzuki–Miyaura cross-coupling showed BCATm inhibitory activity with IC50 = 2.0 μM. A protein crystal structure of 15e revealed that it binds to BCATm within the catalytic site adjacent to the PLP cofactor. The identification of this novel inhibitor series plus the establishment of a BCATm protein structure provided a good starting point for future structure-based discovery of BCATm inhibitors. PMID:26288694

Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis.

PubMed

Anderson, Donald D; Woeller, Collynn F; Chiang, En-Pei; Shane, Barry; Stover, Patrick J

2012-03-02

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.

Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae.

PubMed

Li, Siwei; Ding, Wentao; Zhang, Xueli; Jiang, Huifeng; Bi, Changhao

2016-01-01

Saccharomyces cerevisiae has already been used for heterologous production of fuel chemicals and valuable natural products. The establishment of complicated heterologous biosynthetic pathways in S. cerevisiae became the research focus of Synthetic Biology and Metabolic Engineering. Thus, simple and efficient genomic integration techniques of large number of transcription units are demanded urgently. An efficient DNA assembly and chromosomal integration method was created by combining homologous recombination (HR) in S. cerevisiae and Golden Gate DNA assembly method, designated as modularized two-step (M2S) technique. Two major assembly steps are performed consecutively to integrate multiple transcription units simultaneously. In Step 1, Modularized scaffold containing a head-to-head promoter module and a pair of terminators was assembled with two genes. Thus, two transcription units were assembled with Golden Gate method into one scaffold in one reaction. In Step 2, the two transcription units were mixed with modules of selective markers and integration sites and transformed into S. cerevisiae for assembly and integration. In both steps, universal primers were designed for identification of correct clones. Establishment of a functional β-carotene biosynthetic pathway in S. cerevisiae within 5 days demonstrated high efficiency of this method, and a 10-transcriptional-unit pathway integration illustrated the capacity of this method. Modular design of transcription units and integration elements simplified assembly and integration procedure, and eliminated frequent designing and synthesis of DNA fragments in previous methods. Also, by assembling most parts in Step 1 in vitro, the number of DNA cassettes for homologous integration in Step 2 was significantly reduced. Thus, high assembly efficiency, high integration capacity, and low error rate were achieved.

In vitro cartilage construct generation from silk fibroin- chitosan porous scaffold and umbilical cord blood derived human mesenchymal stem cells in dynamic culture condition.

PubMed

Agrawal, Parinita; Pramanik, Krishna; Biswas, Amit; Ku Patra, Ranjan

2018-02-01

Cartilage construct generation includes a scaffold with appropriate composition to mimic matrix of the damaged tissue on which the stem cells grow and differentiate. In this study, umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) were seeded on freeze dried porous silk-fibroin (SF)/chitosan (CS) scaffolds. Influence of static and dynamic (spinner flask bioreactor) culture conditions on the developing cartilage construct were studied by in-vitro characterization for viability, proliferation, distribution, and chondrogenic differentiation of hMSCs over the scaffold. Constructs developed in spinner flask consisted of 62% live cells, and exhibited 543% more cell density at the core than constructs cultured in static system. Quantification of DNA and glycosaminoglycans accumulation after 21 days showed the progression of chondrogenic differentiation of hMSCs was higher in dynamic culture compared to static one. In constructs generated under dynamic condition, histology staining for proteoglycan matrix, and fluorescence staining for collagen-II and aggrecan showed positive correlation between early and late stage chondrogenic markers, which was further confirmed by quantitative PCR analysis, showing low collagen-I expression and highly expressed Sox9, collagen-II and aggrecan. The present study demonstrated that construct generated by combining 3D SF/CS scaffold with UCB-hMSCs under dynamic condition using spinner flask bioreactor can be used for cartilage tissue regeneration for future medical treatments. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 397-407, 2018. © 2017 Wiley Periodicals, Inc.

Structure of p73 DNA-binding domain tetramer modulates p73 transactivation

PubMed Central

Ethayathulla, Abdul S.; Tse, Pui-Wah; Monti, Paola; Nguyen, Sonha; Inga, Alberto; Fronza, Gilberto; Viadiu, Hector

2012-01-01

The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation. PMID:22474346

DNA rendering of polyhedral meshes at the nanoscale

NASA Astrophysics Data System (ADS)

Benson, Erik; Mohammed, Abdulmelik; Gardell, Johan; Masich, Sergej; Czeizler, Eugen; Orponen, Pekka; Högberg, Björn

2015-07-01

It was suggested more than thirty years ago that Watson-Crick base pairing might be used for the rational design of nanometre-scale structures from nucleic acids. Since then, and especially since the introduction of the origami technique, DNA nanotechnology has enabled increasingly more complex structures. But although general approaches for creating DNA origami polygonal meshes and design software are available, there are still important constraints arising from DNA geometry and sense/antisense pairing, necessitating some manual adjustment during the design process. Here we present a general method of folding arbitrary polygonal digital meshes in DNA that readily produces structures that would be very difficult to realize using previous approaches. The design process is highly automated, using a routeing algorithm based on graph theory and a relaxation simulation that traces scaffold strands through the target structures. Moreover, unlike conventional origami designs built from close-packed helices, our structures have a more open conformation with one helix per edge and are therefore stable under the ionic conditions usually used in biological assays.

DNA rendering of polyhedral meshes at the nanoscale.

PubMed

Benson, Erik; Mohammed, Abdulmelik; Gardell, Johan; Masich, Sergej; Czeizler, Eugen; Orponen, Pekka; Högberg, Björn

2015-07-23

It was suggested more than thirty years ago that Watson-Crick base pairing might be used for the rational design of nanometre-scale structures from nucleic acids. Since then, and especially since the introduction of the origami technique, DNA nanotechnology has enabled increasingly more complex structures. But although general approaches for creating DNA origami polygonal meshes and design software are available, there are still important constraints arising from DNA geometry and sense/antisense pairing, necessitating some manual adjustment during the design process. Here we present a general method of folding arbitrary polygonal digital meshes in DNA that readily produces structures that would be very difficult to realize using previous approaches. The design process is highly automated, using a routeing algorithm based on graph theory and a relaxation simulation that traces scaffold strands through the target structures. Moreover, unlike conventional origami designs built from close-packed helices, our structures have a more open conformation with one helix per edge and are therefore stable under the ionic conditions usually used in biological assays.

Synthesis and characterization of Cu(II)-based anticancer chemotherapeutic agent targeting topoisomerase Iα: in vitro DNA binding, pBR322 cleavage, molecular docking studies and cytotoxicity against human cancer cell lines.

PubMed

Tabassum, Sartaj; Zaki, Mehvash; Afzal, Mohd; Arjmand, Farukh

2014-03-03

New metal-based anticancer chemotherapeutic drug candidates [Cu(phen)L](NO₃)₂ (1) and [Zn(phen)L](NO₃)₂ (2) were synthesized from ligand L (derived from pharmacophore scaffold barbituric acid and pyrazole). In vitro DNA binding studies of the L, 1 and 2 were carried out by various biophysical techniques revealing electrostatic mode. Complex 1 cleaves pBR322 DNA via oxidative pathway and recognizes major groove of DNA double helix. The molecular docking study was carried out to ascertain the mode of action towards the molecular target DNA and enzymes. The complex 1 exhibited remarkably good anticancer activity on a panel of human cancer cell lines (GI₅₀ values < 10 μg/ml), and to elucidate the mechanism of cancer inhibition, Topo-I enzymatic activity was carried out. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Scaffold attachment factor B suppresses HIV-1 infection of CD4+ cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat.

PubMed

Ma, Li; Sun, Li; Jin, Xia; Xiong, Si-Dong; Wang, Jian-Hua

2018-06-10

The 5' end of HIV-1 long terminal repeat (LTR) promoter plays an essential role in driving viral transcription and productive infection. Multiple host and viral factors regulate LTR activity and modulate HIV-1 latency. Manipulation of the HIV-1 LTR provides a potential therapeutic strategy for combating HIV-1 persistence. In this study, we identified an RNA-/DNA-binding protein, Scaffold Attachment Factor B (SAFB1) as a host-cell factor that represses HIV-1 transcription. We found that SAFB1 bound to HIV-1 5`-LTR and significantly repressed 5`-LTR-driven-viral transcription and HIV-1 infection of CD4 + T cells. Mechanistically, SAFB1-mediated repression of HIV-1 transcription and infection was independent of its RNA- and DNA-binding capacities, instead, by binding to phosphorylated RNA polymerase II (RNA pol II), SAFB1 blocked its recruitment to the HIV-1 LTR. Of note, the SAFB1-mediated repression of HIV-1 transcription from proviral DNA maintained HIV-1 latency in CD4 + T cells. In summary, our findings reveal that SAFB1 binds to HIV-1-LTR and physically interacts with phosphorylated RNA pol II, repressing HIV-1 transcription initiation and elongation. Our findings improve the understanding of host modulation of HIV-1 transcription and latency and provide a new host-cell target for improved anti-HIV-1 therapies. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Coverage percentage and raman measurement of cross-tile and scaffold cross-tile based DNA nanostructures.

PubMed

Gnapareddy, Bramaramba; Ahn, Sang Jung; Dugasani, Sreekantha Reddy; Kim, Jang Ah; Amin, Rashid; Mitta, Sekhar Babu; Vellampatti, Srivithya; Kim, Byeonghoon; Kulkarni, Atul; Kim, Taesung; Yun, Kyusik; LaBean, Thomas H; Park, Sung Ha

2015-11-01

We present two free-solution annealed DNA nanostructures consisting of either cross-tile CT1 or CT2. The proposed nanostructures exhibit two distinct structural morphologies, with one-dimensional (1D) nanotubes for CT1 and 2D nanolattices for CT2. When we perform mica-assisted growth annealing with CT1, a dramatic dimensional change occurs where the 1D nanotubes transform into 2D nanolattices due to the presence of the substrate. We assessed the coverage percentage of the 2D nanolattices grown on the mica substrate with CT1 and CT2 as a function of the concentration of the DNA monomer. Furthermore, we fabricated a scaffold cross-tile (SCT), which is a new design of a modified cross-tile that consists of four four-arm junctions with a square aspect ratio. For SCT, eight oligonucleotides are designed in such a way that adjacent strands with sticky ends can produce continuous arms in both the horizontal and vertical directions. The SCT was fabricated via free-solution annealing, and self-assembled SCT produces 2D nanolattices with periodic square cavities. All structures were observed via atomic force microscopy. Finally, we fabricated divalent nickel ion (Ni(2+))- and trivalent dysprosium ion (Dy(3+))-modified 2D nanolattices constructed with CT2 on a quartz substrate, and the ion coordinations were examined via Raman spectroscopy. Copyright © 2015 Elsevier B.V. All rights reserved.

An RNA Origami Octahedron with Intrinsic siRNAs for Potent Gene Knockdown.

PubMed

Høiberg, Hans Christian; Sparvath, Steffen M; Andersen, Veronica L; Kjems, Jørgen; Andersen, Ebbe S

2018-05-26

The fields of DNA and RNA nanotechnology have established nucleic acids as valuable building blocks for functional nanodevices with applications in nanomedicine. Here, a simple method for designing and assembling a 3D scaffolded RNA origami wireframe structure with intrinsic functioning small interfering RNAs (siRNAs) embedded is introduced. Uniquely, the method uses an mRNA fragment as scaffold strand, which is folded by sequence-complementarity of nine shorter synthetic strands. High-yield production of the intended 3D structure is verified by transmission electron microscopy (TEM). Production of functional siRNAs is facilitated by incorporating recognition sites for Dicer at selected locations in the structure, and efficient silencing of a target reporter gene is demonstrated. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

PubMed

Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

2011-12-09

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

Decellularized and matured esophageal scaffold for circumferential esophagus replacement: Proof of concept in a pig model.

PubMed

Luc, Guillaume; Charles, Guillaume; Gronnier, Caroline; Cabau, Magali; Kalisky, Charlotte; Meulle, Mallory; Bareille, Reine; Roques, Samantha; Couraud, Lionel; Rannou, Johanna; Bordenave, Laurence; Collet, Denis; Durand, Marlène

2018-08-01

Surgical resection of the esophagus requires sacrificing a long portion of it. Its replacement by the demanding gastric pull-up or colonic interposition techniques may be avoided by using short biologic scaffolds composed of decellularized matrix (DM). The aim of this study was to prepare, characterize, and assess the in vivo remodeling of DM and its clinical impact in a preclinical model. A dynamic chemical and enzymatic decellularization protocol of porcine esophagus was set up and optimized. The resulting DM was mechanically and biologically characterized by DNA quantification, histology, and histomorphometry techniques. Then, in vitro and in vivo tests were performed, such as DM recellularization with human or porcine adipose-derived stem cells, or porcine stromal vascular fraction, and maturation in rat omentum. Finally, the DM, matured or not, was implanted as a 5-cm-long esophagus substitute in an esophagectomized pig model. The developed protocol for esophageal DM fulfilled previously established criteria of decellularization and resulted in a scaffold that maintained important biologic components and an ultrastructure consistent with a basement membrane complex. In vivo implantation was compatible with life without major clinical complications. The DM's scaffold in vitro characteristics and in vivo implantation showed a pattern of constructive remodeling mimicking major native esophageal characteristics. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative analysis of poly-glycolic acid-based hybrid polymer starter matrices for in vitro tissue engineering.

PubMed

Generali, Melanie; Kehl, Debora; Capulli, Andrew K; Parker, Kevin K; Hoerstrup, Simon P; Weber, Benedikt

2017-10-01

Biodegradable scaffold matrixes form the basis of any in vitro tissue engineering approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until the scaffold is replaced by neo-tissue. In this context several synthetic polymers have been investigated, however a concise systematic comparative analyses is missing. Therefore, the present study systematically compares three frequently used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA) under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymers structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGA-poly-4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly-caprolactone (PCL) patches. These patches were analyzed after 21days of culture qualitative by histology and quantitative by determining the amount of DNA, glycosaminoglycan and hydroxyproline. We found that PGA-P4HB and PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds (p<0.05). Polymer remnants were visualized by polarization microscopy. In addition, biomechanical properties of the tissue engineered patches were determined in comparison to native tissue. This study may allow future studies to specifically select certain polymer starter matrices aiming at specific tissue properties of the bioengineered constructs in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2009-01-01

DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14–18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment. PMID:19276204

Androgen actions in mouse wound healing: Minimal in vivo effects of local antiandrogen delivery.

PubMed

Wang, Yiwei; Simanainen, Ulla; Cheer, Kenny; Suarez, Francia G; Gao, Yan Ru; Li, Zhe; Handelsman, David; Maitz, Peter

2016-05-01

The aims of this work were to define the role of androgens in female wound healing and to develop and characterize a novel wound dressing with antiandrogens. Androgens retard wound healing in males, but their role in female wound healing has not been established. To understand androgen receptor (AR)-mediated androgen actions in male and female wound healing, we utilized the global AR knockout (ARKO) mouse model, with a mutated AR deleting the second zinc finger to disrupt DNA binding and transcriptional activation. AR inactivation enhanced wound healing rate in males by increasing re-epithelialization and collagen deposition even when wound contraction was eliminated. Cell proliferation and migration in ARKO male fibroblasts was significantly increased compared with wild-type (WT) fibroblasts. However, ARKO females showed a similar healing rate compared to WT females. To exploit local antiandrogen effects in wound healing, while minimizing off-target systemic effects, we developed a novel electrospun polycaprolactone (PCL) scaffold wound dressing material for sustained local antiandrogen delivery. Using the antiandrogen hydroxyl flutamide (HF) at 1, 5, and 10 mg/mL in PCL scaffolds, controlled HF delivery over 21 days significantly enhanced in vitro cell proliferation of human dermal fibroblasts and human keratinocytes. HF-PCL scaffolds also promoted in vivo wound healing in mice compared with open wounds but not to PCL scaffolds. © 2016 by the Wound Healing Society.

Theory and modeling of particles with DNA-mediated interactions

NASA Astrophysics Data System (ADS)

Licata, Nicholas A.

2008-05-01

In recent years significant attention has been attracted to proposals which utilize DNA for nanotechnological applications. Potential applications of these ideas range from the programmable self-assembly of colloidal crystals, to biosensors and nanoparticle based drug delivery platforms. In Chapter I we introduce the system, which generically consists of colloidal particles functionalized with specially designed DNA markers. The sequence of bases on the DNA markers determines the particle type. Due to the hybridization between complementary single-stranded DNA, specific, type-dependent interactions can be introduced between particles by choosing the appropriate DNA marker sequences. In Chapter II we develop a statistical mechanical description of the aggregation and melting behavior of particles with DNA-mediated interactions. In Chapter III a model is proposed to describe the dynamical departure and diffusion of particles which form reversible key-lock connections. In Chapter IV we propose a method to self-assemble nanoparticle clusters using DNA scaffolds. A natural extension is discussed in Chapter V, the programmable self-assembly of nanoparticle clusters where the desired cluster geometry is encoded using DNA-mediated interactions. In Chapter VI we consider a nanoparticle based drug delivery platform for targeted, cell specific chemotherapy. In Chapter VII we present prospects for future research: the connection between DNA-mediated colloidal crystallization and jamming, and the inverse problem in self-assembly.

Stability of DNA Origami Nanoarrays in Cell Lysate

PubMed Central

Mei, Qian; Wei, Xixi; Su, Fengyu; Liu, Yan; Youngbull, Cody; Johnson, Roger; Lindsay, Stuart; Yan, Hao; Meldrum, Deirdre

2012-01-01

Scaffolded DNA origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the origami–lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms. PMID:21366226

The scaffold protein Nde1 safeguards the brain genome during S phase of early neural progenitor differentiation

PubMed Central

Houlihan, Shauna L; Feng, Yuanyi

2014-01-01

Successfully completing the S phase of each cell cycle ensures genome integrity. Impediment of DNA replication can lead to DNA damage and genomic disorders. In this study, we show a novel function for NDE1, whose mutations cause brain developmental disorders, in safeguarding the genome through S phase during early steps of neural progenitor fate restrictive differentiation. Nde1 mutant neural progenitors showed catastrophic DNA double strand breaks concurrent with the DNA replication. This evoked DNA damage responses, led to the activation of p53-dependent apoptosis, and resulted in the reduction of neurons in cortical layer II/III. We discovered a nuclear pool of Nde1, identified the interaction of Nde1 with cohesin and its associated chromatin remodeler, and showed that stalled DNA replication in Nde1 mutants specifically occurred in mid-late S phase at heterochromatin domains. These findings suggest that NDE1-mediated heterochromatin replication is indispensible for neuronal differentiation, and that the loss of NDE1 function may lead to genomic neurological disorders. DOI: http://dx.doi.org/10.7554/eLife.03297.001 PMID:25245017

DNA origami nanorobot fiber optic genosensor to TMV.

PubMed

Torelli, Emanuela; Manzano, Marisa; Srivastava, Sachin K; Marks, Robert S

2018-01-15

In the quest of greater sensitivity and specificity of diagnostic systems, one continually searches for alternative DNA hybridization methods, enabling greater versatility and where possible field-enabled detection of target analytes. We present, herein, a hybrid molecular self-assembled scaffolded DNA origami entity, intimately immobilized via capture probes linked to aminopropyltriethoxysilane, onto a glass optical fiber end-face transducer, thus producing a novel biosensor. Immobilized DNA nanorobots with a switchable flap can then be actuated by a specific target DNA present in a sample, by exposing a hemin/G-quadruplex DNAzyme, which then catalyzes the generation of chemiluminescence, once the specific fiber probes are immersed in a luminol-based solution. Integrating organic nanorobots to inorganic fiber optics creates a hybrid system that we demonstrate as a proof-of-principle can be utilized in specific DNA sequence detection. This system has potential applications in a wide range of fields, including point-of-care diagnostics or cellular in vivo biosensing when using ultrathin fiber optic probes for research purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

PubMed Central

Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

2015-01-01

Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799

DNA-Mediated Patterning of Single Quantum Dot Nanoarrays: A Reusable Platform for Single-Molecule Control

NASA Astrophysics Data System (ADS)

Huang, Da; Freeley, Mark; Palma, Matteo

2017-03-01

We present a facile strategy of general applicability for the assembly of individual nanoscale moieties in array configurations with single-molecule control. Combining the programming ability of DNA as a scaffolding material with a one-step lithographic process, we demonstrate the patterning of single quantum dots (QDs) at predefined locations on silicon and transparent glass surfaces: as proof of concept, clusters of either one, two, or three QDs were assembled in highly uniform arrays with a 60 nm interdot spacing within each cluster. Notably, the platform developed is reusable after a simple cleaning process and can be designed to exhibit different geometrical arrangements.

Nondestructive Methods for Monitoring Cell Removal During Rat Liver Decellularization.

PubMed

Geerts, Sharon; Ozer, Sinan; Jaramillo, Maria; Yarmush, Martin L; Uygun, Basak E

2016-07-01

Whole liver engineering holds the promise to create transplantable liver grafts that may serve as substitutes for donor organs, addressing the donor shortage in liver transplantation. While decellularization and recellularization of livers in animal models have been successfully achieved, scale up to human livers has been slow. There are a number of donor human livers that are discarded because they are not found suitable for transplantation, but are available for engineering liver grafts. These livers are rejected due to a variety of reasons, which in turn may affect the decellularization outcome. Hence, a one-size-fit-for all decellularization protocol may not result in scaffolds with consistent matrix quality, subsequently influencing downstream recellularization and transplantation outcomes. There is a need for a noninvasive monitoring method to evaluate the extent of cell removal, while ensuring preservation of matrix components during decellularization. In this study, we decellularized rat livers using a protocol previously established by our group, and we monitored decellularization through traditional destructive techniques, including evaluation of DNA, collagen, and glycosaminoglycan (GAG) content in decellularized scaffolds, as well as histology. In addition, we used computed tomography and perfusate analysis as alternative nondestructive decellularization monitoring methods. We found that DNA removal correlates well with the Hounsfield unit of the liver, and perfusate analysis revealed that significant amount of GAG is removed during perfusion with 0.1% sodium dodecyl sulfate. This allowed for optimization of our decellularization protocol leading to scaffolds that have significantly higher GAG content, while maintaining appropriate removal of cellular contents. The significance of this is the creation of a nondestructive monitoring strategy that can be used for optimization of decellularization protocols for individual human livers available for liver engineering.

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

PubMed

Hilz, Florian M; Ahrens, Philipp; Grad, Sibylle; Stoddart, Martin J; Dahmani, Chiheb; Wilken, Frauke L; Sauerschnig, Martin; Niemeyer, Philipp; Zwingmann, Jörn; Burgkart, Rainer; von Eisenhart-Rothe, Rüdiger; Südkamp, Norbert P; Weyh, Thomas; Imhoff, Andreas B; Alini, Mauro; Salzmann, Gian M

2014-02-01

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production. © 2013 Wiley Periodicals, Inc.

Nondestructive Methods for Monitoring Cell Removal During Rat Liver Decellularization

PubMed Central

Geerts, Sharon; Ozer, Sinan; Jaramillo, Maria; Yarmush, Martin L.

2016-01-01

Whole liver engineering holds the promise to create transplantable liver grafts that may serve as substitutes for donor organs, addressing the donor shortage in liver transplantation. While decellularization and recellularization of livers in animal models have been successfully achieved, scale up to human livers has been slow. There are a number of donor human livers that are discarded because they are not found suitable for transplantation, but are available for engineering liver grafts. These livers are rejected due to a variety of reasons, which in turn may affect the decellularization outcome. Hence, a one-size-fit-for all decellularization protocol may not result in scaffolds with consistent matrix quality, subsequently influencing downstream recellularization and transplantation outcomes. There is a need for a noninvasive monitoring method to evaluate the extent of cell removal, while ensuring preservation of matrix components during decellularization. In this study, we decellularized rat livers using a protocol previously established by our group, and we monitored decellularization through traditional destructive techniques, including evaluation of DNA, collagen, and glycosaminoglycan (GAG) content in decellularized scaffolds, as well as histology. In addition, we used computed tomography and perfusate analysis as alternative nondestructive decellularization monitoring methods. We found that DNA removal correlates well with the Hounsfield unit of the liver, and perfusate analysis revealed that significant amount of GAG is removed during perfusion with 0.1% sodium dodecyl sulfate. This allowed for optimization of our decellularization protocol leading to scaffolds that have significantly higher GAG content, while maintaining appropriate removal of cellular contents. The significance of this is the creation of a nondestructive monitoring strategy that can be used for optimization of decellularization protocols for individual human livers available for liver engineering. PMID:27169332

Modification of Rat Lung Decellularization Protocol Based on Dynamic Conductometry of Working Solution.

PubMed

Kuevda, E V; Gubareva, E A; Gumenyuk, I S; Sotnichenko, A S; Gilevich, I V; Nakokhov, R Z; Rusinova, T V; Yudina, T G; Red'ko, A N; Alekseenko, S N

2017-03-01

We modified the protocol of obtaining of biological scaffolds of rat lungs based on dynamic recording of specific resistivity of working detergent solution (conductometry) during perfusion decellularization. Termination of sodium deoxycholate exposure after attaining ionic equilibrium plateau did not impair the quality of decellularization and preserved structural matrix components, which was confirmed by morphological analysis and quantitative assay of residual DNA.

The Importance of Connexin Hemichannels During Chondroprogenitor Cell Differentiation in Hydrogel Versus Microtissue Culture Models

PubMed Central

Schrobback, Karsten; Klein, Travis Jacob

2015-01-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell–cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell–cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5′-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell–cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs. PMID:25693425

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models.

PubMed

Schrobback, Karsten; Klein, Travis Jacob; Woodfield, Tim B F

2015-06-01

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.

PubMed

Ramsay, Nicola; Jemth, Ann-Sofie; Brown, Anthony; Crampton, Neal; Dear, Paul; Holliger, Philipp

2010-04-14

DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting "CyDNA" displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties.

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.

PubMed

Flaibani, Marina; Luni, Camilla; Sbalchiero, Elisa; Elvassore, Nicola

2009-01-01

It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library.

PubMed

Traxlmayr, Michael W; Kiefer, Jonathan D; Srinivas, Raja R; Lobner, Elisabeth; Tisdale, Alison W; Mehta, Naveen K; Yang, Nicole J; Tidor, Bruce; Wittrup, K Dane

2016-10-21

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T m of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

PubMed

Rao, Harita; Damian, Mariana S; Alshiekh, Alak; Elmroth, Sofi K C; Diederichsen, Ulf

2015-12-28

Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues.

Molecular threading and tunable molecular recognition on DNA origami nanostructures.

PubMed

Wu, Na; Czajkowsky, Daniel M; Zhang, Jinjin; Qu, Jianxun; Ye, Ming; Zeng, Dongdong; Zhou, Xingfei; Hu, Jun; Shao, Zhifeng; Li, Bin; Fan, Chunhai

2013-08-21

The DNA origami technology holds great promise for the assembly of nanoscopic technological devices and studies of biochemical reactions at the single-molecule level. For these, it is essential to establish well controlled attachment of functional materials to predefined sites on the DNA origami nanostructures for reliable measurements and versatile applications. However, the two-sided nature of the origami scaffold has shown limitations in this regard. We hypothesized that holes of the commonly used two-dimensional DNA origami designs are large enough for the passage of single-stranded (ss)-DNA. Sufficiently long ssDNA initially located on one side of the origami should thus be able to "thread" to the other side through the holes in the origami sheet. By using an origami sheet attached with patterned biotinylated ssDNA spacers and monitoring streptavidin binding with atomic force microscopic (AFM) imaging, we provide unambiguous evidence that the biotin ligands positioned on one side have indeed threaded through to the other side. Our finding reveals a previously overlooked critical design feature that should provide new interpretations to previous experiments and new opportunities for the construction of origami structures with new functional capabilities.

Modular structural elements in the replication origin region of Tetrahymena rDNA.

PubMed Central

Du, C; Sanzgiri, R P; Shaiu, W L; Choi, J K; Hou, Z; Benbow, R M; Dobbs, D L

1995-01-01

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication. Images PMID:7784181

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

PubMed Central

Churchill, M E; Jones, D N; Glaser, T; Hefner, H; Searles, M A; Travers, A A

1995-01-01

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region. Images PMID:7720717

Pre-treatment of synthetic elastomeric scaffolds by cardiac fibroblasts improves engineered heart tissue

PubMed Central

Radisic, Milica; Park, Hyoungshin; Martens, Timothy P.; Salazar-Lazaro, Johanna E.; Geng, Wenliang; Wang, Yadong; Langer, Robert; Freed, Lisa E.; Vunjak-Novakovic, Gordana

2009-01-01

Native myocardium consists of several cell types, of which approximately one-third are myocytes and most of the nonmyocytes are fibroblasts. By analogy with monolayer culture in which fibroblasts were removed to prevent overgrowth, early attempts to engineer myocardium utilized cell populations enriched for cardiac myocytes (CMs; ~80–90% of total cells). We hypothesized that the pre-treatment of synthetic elastomeric scaffolds with cardiac fibroblasts (CFs) will enhance the functional assembly of the engineered cardiac constructs by creating an environment supportive of cardiomyocyte attachment and function. Cells isolated from neonatal rat ventricles were prepared to form three distinct populations: rapidly plating cells identified as CFs, slowly plating cells identified as CMs, and unseparated initial population of cells (US). The cell fractions (3 × 106 cells total) were seeded into poly(glycerol sebacate) scaffolds (highly porous discs, 5 mm in diameter × 2-mm thick) using Matrigel™, either separately (CM or CF), concurrently (US), or sequentially (CF pre-treatment followed by CM culture, CF + CM), and cultured in spinner flasks. The CF + CM group had the highest amplitude of contraction and the lowest excitation threshold, superior DNA content, and higher glucose consumption rate. The CF + CM group exhibited compact 100- to 200-μm thick layers of elongated myocytes aligned in parallel over layers of collagen-producing fibroblasts, while US and CM groups exhibited scattered and poorly elongated myocytes. The sequential co-culture of CF and CM on a synthetic elastomer scaffold thus created an environment supportive of cardiomyocyte attachment, differentiation, and contractile function, presumably due to scaffold conditioning by cultured fibroblasts. When implanted over the infarcted myocardium in a nude rat model, cell-free poly(glycerol sebacate) remained at the ventricular wall after 2 weeks of in vivo, and was vascularized. PMID:18041719

CyDNA: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase

PubMed Central

2010-01-01

DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting “CyDNA” displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties. PMID:20235594

Structure of transcription factor HetR required for heterocyst differentiation in cyanobacteria

PubMed Central

Kim, Youngchang; Joachimiak, Grazyna; Ye, Zi; Binkowski, T. Andrew; Zhang, Rongguang; Gornicki, Piotr; Callahan, Sean M.; Hess, Wolfgang R.; Haselkorn, Robert; Joachimiak, Andrzej

2011-01-01

HetR is an essential regulator of heterocyst development in cyanobacteria. HetR binds to a DNA palindrome upstream of the hetP gene. We report the crystal structure of HetR from Fischerella at 3.0 Å. The protein is a dimer comprised of a central DNA-binding unit containing the N-terminal regions of the two subunits organized with two helix-turn-helix motifs; two globular flaps extending in opposite directions; and a hood over the central core formed from the C-terminal subdomains. The flaps and hood have no structural precedent in the protein database, therefore representing new folds. The structural assignments are supported by site-directed mutagenesis and DNA-binding studies. We suggest that HetR serves as a scaffold for assembly of transcription components critical for heterocyst development. PMID:21628585

Structural and Thermodynamic Signatures of DNA Recognition by Mycobacterium tuberculosis DnaA

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

Tsodikov, Oleg V.; Biswas, Tapan

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 {angstrom} resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 {angstrom}). Thesemore » structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.« less

Global Gene Expression Analysis of Yeast Cells during Sake Brewing▿ †

PubMed Central

Wu, Hong; Zheng, Xiaohong; Araki, Yoshio; Sahara, Hiroshi; Takagi, Hiroshi; Shimoi, Hitoshi

2006-01-01

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process. PMID:16997994

Optical mapping and its potential for large-scale sequencing projects.

PubMed

Aston, C; Mishra, B; Schwartz, D C

1999-07-01

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.

Nanostructured 3D constructs based on chitosan and chondroitin sulphate multilayers for cartilage tissue engineering.

PubMed

Silva, Joana M; Georgi, Nicole; Costa, Rui; Sher, Praveen; Reis, Rui L; Van Blitterswijk, Clemens A; Karperien, Marcel; Mano, João F

2013-01-01

Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs.

ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure

PubMed Central

1994-01-01

Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II. PMID:7929577

Sequencing and assembly of the 22-gb loblolly pine genome.

PubMed

Zimin, Aleksey; Stevens, Kristian A; Crepeau, Marc W; Holtz-Morris, Ann; Koriabine, Maxim; Marçais, Guillaume; Puiu, Daniela; Roberts, Michael; Wegrzyn, Jill L; de Jong, Pieter J; Neale, David B; Salzberg, Steven L; Yorke, James A; Langley, Charles H

2014-03-01

Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer "super-reads," rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.

Programmed self-assembly of DNA/RNA for biomedical applications

NASA Astrophysics Data System (ADS)

Wang, Pengfei

Three self-assembly strategies were utilized for assembly of novel functional DNA/RNA nanostructures. RNA-DNA hybrid origami method was developed to fabricate nano-objects (ribbon, rectangle, and triangle) with precisely controlled geometry. Unlike conventional DNA origami which use long DNA single strand as scaffold, a long RNA single strand was used instead, which was folded by short DNA single strands (staples) into prescribed objects through sequence specific hybridization between RNA and DNA. Single stranded tiles (SST) and RNA-DNA hybrid origami were utilized to fabricate a variety of barcode-like nanostructures with unique patterns by expanding a plain rectangle via introducing spacers (10-bp dsDNA segment) between parallel duplexes. Finally, complex 2D array and 3D polyhedrons with multiple patterns within one structure were assembled from simple DNA motifs. Two demonstrations of biomedical applications of DNA nanotechnology were presented. Firstly, lambda-DNA was used as template to direct the fabrication of multi-component magnetic nanoparticle chains. Nuclear magnetic relaxation (NMR) characterization showed superb magnetic relaxativity of the nanoparticle chains which have large potential to be utilized as MRI contrast agents. Secondly, DNA nanotechnology was introduced into the conformational study of a routinely used catalytic DNAzyme, the RNA-cleaving 10-23 DNAzyme. The relative angle between two flanking duplexes of the catalytic core was determined (94.8°), which shall be able to provide a clue to further understanding of the cleaving mechanism of this DNAzyme from a conformational perspective.

Structural Transformation of Wireframe DNA Origami via DNA Polymerase Assisted Gap-Filling.

PubMed

Agarwal, Nayan P; Matthies, Michael; Joffroy, Bastian; Schmidt, Thorsten L

2018-03-27

The programmability of DNA enables constructing nanostructures with almost any arbitrary shape, which can be decorated with many functional materials. Moreover, dynamic structures can be realized such as molecular motors and walkers. In this work, we have explored the possibility to synthesize the complementary sequences to single-stranded gap regions in the DNA origami scaffold cost effectively by a DNA polymerase rather than by a DNA synthesizer. For this purpose, four different wireframe DNA origami structures were designed to have single-stranded gap regions. This reduced the number of staple strands needed to determine the shape and size of the final structure after gap filling. For this, several DNA polymerases and single-stranded binding (SSB) proteins were tested, with T4 DNA polymerase being the best fit. The structures could be folded in as little as 6 min, and the subsequent optimized gap-filling reaction was completed in less than 3 min. The introduction of flexible gap regions results in fully collapsed or partially bent structures due to entropic spring effects. Finally, we demonstrated structural transformations of such deformed wireframe DNA origami structures with DNA polymerases including the expansion of collapsed structures and the straightening of curved tubes. We anticipate that this approach will become a powerful tool to build DNA wireframe structures more material-efficiently, and to quickly prototype and test new wireframe designs that can be expanded, rigidified, or mechanically switched. Mechanical force generation and structural transitions will enable applications in structural DNA nanotechnology, plasmonics, or single-molecule biophysics.

TopBP1-mediated DNA processing during mitosis.

PubMed

Gallina, Irene; Christiansen, Signe Korbo; Pedersen, Rune Troelsgaard; Lisby, Michael; Oestergaard, Vibe H

2016-01-01

Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

The shear flow processing of controlled DNA tethering and stretching for organic molecular electronics.

PubMed

Yu, Guihua; Kushwaha, Amit; Lee, Jungkyu K; Shaqfeh, Eric S G; Bao, Zhenan

2011-01-25

DNA has been recently explored as a powerful tool for developing molecular scaffolds for making reproducible and reliable metal contacts to single organic semiconducting molecules. A critical step in the process of exploiting DNA-organic molecule-DNA (DOD) array structures is the controlled tethering and stretching of DNA molecules. Here we report the development of reproducible surface chemistry for tethering DNA molecules at tunable density and demonstrate shear flow processing as a rationally controlled approach for stretching/aligning DNA molecules of various lengths. Through enzymatic cleavage of λ-phage DNA to yield a series of DNA chains of various lengths from 17.3 μm down to 4.2 μm, we have investigated the flow/extension behavior of these tethered DNA molecules under different flow strengths in the flow-gradient plane. We compared Brownian dynamic simulations for the flow dynamics of tethered λ-DNA in shear, and found our flow-gradient plane experimental results matched well with our bead-spring simulations. The shear flow processing demonstrated in our studies represents a controllable approach for tethering and stretching DNA molecules of various lengths. Together with further metallization of DNA chains within DOD structures, this bottom-up approach can potentially enable efficient and reliable fabrication of large-scale nanoelectronic devices based on single organic molecules, therefore opening opportunities in both fundamental understanding of charge transport at the single molecular level and many exciting applications for ever-shrinking molecular circuits.

New modulated design and synthesis of chiral CuII/SnIV bimetallic potential anticancer drug entity: In vitro DNA binding and pBR322 DNA cleavage activity

NASA Astrophysics Data System (ADS)

Tabassum, Sartaj; Sharma, Girish Chandra; Arjmand, Farukh

2012-05-01

A new chiral ligand scaffold L derived from (R)-2-amino-2-phenyl ethanol and diethyl oxalate was isolated and thoroughly characterized by various spectroscopic methods. The ligand L was allowed to react with CuCl2·2H2O and NiCl2·6H2O to achieve monometallic complexes 1 and 2, respectively. Subsequently modulation of 1 and 2 was carried out in the presence of SnCl4·5H2O to obtain heterobimetallic potential drug candidates 3 and 4 possessing (CuII/SnIV and NiII/SnIV) metallic cores, respectively and characterized by elemental analysis and spectroscopic data including 1H, 13C and 119Sn NMR in case of 3 and 4. In vitro DNA binding studies revealed that complex 3 avidly binds to DNA as quantified by Kb and Ksv values. Complex 3 exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322 DNA and the cleavage activity of 3 was significantly enhanced in the presence of activators and follows the order H2O2 > Asc > MPA > GSH. Complex 3 cleave pBR322 DNA via hydrolytic pathway and accessible to major groove of DNA.

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis

PubMed Central

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura

2015-01-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. PMID:25986610

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

PubMed Central

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P.H.

2014-01-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. PMID:24609384

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture.

PubMed

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P H

2014-05-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

BuD, a helix–loop–helix DNA-binding domain for genome modification

PubMed Central

Stella, Stefano; Molina, Rafael; López-Méndez, Blanca; Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza; Campos-Olivas, Ramon; Duchateau, Phillippe; Montoya, Guillermo

2014-01-01

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing. PMID:25004980

Comparative Proteomic Analyses of Human Adipose Extracellular Matrices Decellularized Using Alternative Procedures.

PubMed

Thomas-Porch, Caasy; Li, Jie; Zanata, Fabiana; Martin, Elizabeth C; Pashos, Nicholas; Genemaras, Kaylynn; Poche, J Nicholas; Totaro, Nicholas P; Bratton, Melyssa R; Gaupp, Dina; Frazier, Trivia; Wu, Xiying; Ferreira, Lydia Masako; Tian, Weidong; Wang, Guangdi; Bunnell, Bruce A; Flynn, Lauren; Hayes, Daniel; Gimble, Jeffrey M

2018-04-25

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for DNA depletion, ECM composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy (SEM). Mass spectrometry (MS) based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP + transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

3D Printing of Human Tissue Mimics via Layer-by-Layer Assembly of Polymer/Hydrogel Biopapers

NASA Astrophysics Data System (ADS)

Ringeisen, Bradley

2015-03-01

The foundations of tissue engineering were built on two fundamental areas of research: cells and scaffolds. Multipotent cells and their derivatives are traditionally randomly seeded into sophisticated polymer or hydrogel scaffolds, ultimately with the goal of forming a tissue-like material through cell differentiation and cell-material interactions. One problem with this approach is that no matter how complex or biomimetic the scaffold is, the cells are still homogeneously distributed throughout this three dimensional (3D) material. Natural tissue is inherently heterogeneous on both a microscopic and macroscopic level. It also contains different types of cells in close proximity, extracellular matrix, voids, and a complex vascularized network. Recently developed 3D cell and organ printers may be able to enhance traditional tissue engineering experiments by building scaffolds layer-by-layer that are crafted to mimic the microscopic and macroscopic structure of natural tissue or organs. Over the past decade, my laboratory has developed a capillary-free, live cell printer termed biological laser printing, or BioLP. We find that printed cells do not express heat shock protein and retain >99% viability. Printed cells also incur no DNA strand fracture and preserve their ability to differentiate. Recent work has used a layer-by-layer approach, stacking sheets of hybrid polymer/hydrogel biopapers in conjunction with live cell printing to create 3D tissue structures. Our specific work is now focused on the blood-brain-barrier and air-lung interface and will be described during the presentation.

Simultaneous Drug Targeting of the Promoter MYC G-Quadruplex and BCL2 i-Motif in Diffuse Large B-Cell Lymphoma Delays Tumor Growth.

PubMed

Kendrick, Samantha; Muranyi, Andrea; Gokhale, Vijay; Hurley, Laurence H; Rimsza, Lisa M

2017-08-10

Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

NASA Astrophysics Data System (ADS)

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

PubMed

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

DNA Assembly Techniques for Next Generation Combinatorial Biosynthesis of Natural Products

PubMed Central

Cobb, Ryan E.; Ning, Jonathan C.; Zhao, Huimin

2013-01-01

Natural product scaffolds remain important leads for pharmaceutical development. However, transforming a natural product into a drug entity often requires derivatization to enhance the compound’s therapeutic properties. A powerful method by which to perform this derivatization is combinatorial biosynthesis, the manipulation of the genes in the corresponding pathway to divert synthesis towards novel derivatives. While these manipulations have traditionally been carried out via restriction digestion/ligation-based cloning, the shortcomings of such techniques limit their throughput and thus the scope of corresponding combinatorial biosynthesis experiments. In the burgeoning field of synthetic biology, the demand for facile DNA assembly techniques has promoted the development of a host of novel DNA assembly strategies. Here we describe the advantages of these recently-developed tools for rapid, efficient synthesis of large DNA constructs. We also discuss their potential to facilitate the simultaneous assembly of complete libraries of natural product biosynthetic pathways, ushering in the next generation of combinatorial biosynthesis. PMID:24127070

A new fluorescence turn-on nanobiosensor for the detection of micro-RNA-21 based on a DNA-gold nanocluster

NASA Astrophysics Data System (ADS)

Hosseini, Morteza; Ahmadi, Elnaz; Borghei, Yasaman-Sadat; Ganjali, Mohammad Reza

2017-03-01

In this study, DNA/gold nanoclusters (AuNCs) were used to develop an AuNC-based turn-on fluorescence probe for the analysis of mi-RNA-21, which is a potential screening biomarker for cancer detection. AuNCs on a DNA scaffold were prepared through a one-pot wet-chemical route and evaluated by transmission electron microscopy and dynamic light scattering. Experiments revealed that the fluorescence intensity of the DNA-AuNCs showed a gradual increase with the addition of the target species in a concentration range from 1pM to 10 nM. The method had a detection limit of 0.7 pM and was able to discriminate the target species from mismatched mi-RNAs very efficiently. The method was used for the determination of mi-RNA spiked human plasma samples, and was evaluated as a promising nanobiosensor for application in the selective detection of mi-RNA in various biomedical and clinical tests.

DNA Dendrimer: An Efficient Nanocarrier of Functional Nucleic Acids for Intracellular Molecular Sensing

PubMed Central

2015-01-01

Functional nucleic acid (FNA)-based sensing systems have been developed for efficient detection of a wide range of biorelated analytes by employing DNAzymes or aptamers as recognition units. However, their intracellular delivery has always been a concern, mainly in delivery efficiency, kinetics, and the amount of delivered FNAs. Here we report a DNA dendrimer scaffold as an efficient nanocarrier to deliver FNAs and to conduct in situ monitoring of biological molecules in living cells. A histidine-dependent DNAzyme and an anti-ATP aptamer were chosen separately as the model FNAs to make the FNA dendrimer. The FNA-embedded DNA dendrimers maintained the catalytic activity of the DNAzyme or the aptamer recognition function toward ATP in the cellular environment, with no change in sensitivity or specificity. Moreover, these DNA dendrimeric nanocarriers show excellent biocompatibility, high intracellular delivery efficiency, and sufficient stability in a cellular environment. This FNA dendrimeric nanocarrier may find a broad spectrum of applications in biomedical diagnosis and therapy. PMID:24806614

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

PubMed

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

PCNA appears in two populations of slow and fast diffusion with a constant ratio throughout S-phase in replicating mammalian cells.

PubMed

Zessin, Patrick J M; Sporbert, Anje; Heilemann, Mike

2016-01-13

DNA replication is a fundamental cellular process that precedes cell division. Proliferating cell nuclear antigen (PCNA) is a central scaffold protein that orchestrates DNA replication by recruiting many factors essential for the replication machinery. We studied the mobility of PCNA in live mammalian cells using single-particle tracking in combination with photoactivated-localization microscopy (sptPALM) and found two populations. The first population which is only present in cells with active DNA replication, showed slow diffusion and was found to be located in replication foci. The second population showed fast diffusion, and represents the nucleoplasmic pool of unbound PCNA not involved in DNA replication. The ratio of these two populations remained constant throughout different stages of S-phase. A fraction of molecules in both populations showed spatially constrained mobility. We determined an exploration radius of ~100 nm for 13% of the slow-diffusing PCNA molecules, and of ~600 nm for 46% of the fast-diffusing PCNA molecules.

A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters.

PubMed

Hosseini, Morteza; Mehrabi, Fatemeh; Ganjali, Mohammad Reza; Norouzi, Parviz

2016-11-01

A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer-templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA-AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as-prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA-AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Thiaflavan scavenges radicals and inhibits DNA oxidation: a story from the ferrocene modification.

PubMed

Lai, Hai-Wang; Liu, Zai-Qun

2014-06-23

4-Thiaflavan is a sulfur-substituted flavonoid with a benzoxathiin scaffold. The aim of this work is to compare abilities of sulfur and oxygen atom, hydroxyl groups, and ferrocene moiety at different positions of 4-thiaflavan to trap radicals and to inhibit DNA oxidation. It is found that abilities of thiaflavans to trap radicals and to inhibit DNA oxidation are increased in the presence of ferrocene moiety and are further improved by the electron-donating group attaching to thiaflavan skeleton. It can be concluded that the ferrocene moiety plays the major role for thiaflavans to be antioxidants even in the absence of phenolic hydroxyl groups. On the other hand, the antioxidant effectiveness of phenolic hydroxyl groups in thiaflavans can be improved by the electron-donating group. The influences of sulfur and oxygen atoms in thiaflavans on the antioxidant property of para-hydroxyl group exhibit different manners when the thiaflavans are used to trap radicals and to inhibit DNA oxidation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Rapidly evolving homing CRISPR barcodes

PubMed Central

Kalhor, Reza; Mali, Prashant; Church, George M.

2017-01-01

We present here an approach for engineering evolving DNA barcodes in living cells. The methodology entails using a homing guide RNA (hgRNA) scaffold that directs the Cas9-hgRNA complex to target the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show the barcode RNAs can be assayed as single molecules in situ . This integrated approach will have wide ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping. PMID:27918539

Toward DNA electrochemical sensing by free-standing ZnO nanosheets grown on 2D thin-layered MoS2.

PubMed

Yang, Tao; Chen, Meijing; Kong, Qianqian; Luo, Xiliang; Jiao, Kui

2017-03-15

Very recently, the 2-dimensional MoS 2 layer as base substrate integrated with other materials has caused people's emerging attention. In this paper, a thin-layered MoS 2 was prepared through an ultrasonic exfoliation method from bulk MoS 2 and then the free-standing ZnO nanosheet was electrodeposited on the MoS 2 scaffold for DNA sensing. The ZnO/MoS 2 nanocomposite revealed smooth and vertical nanosheets morphology by scanning electron microscopy, compared with the sole MoS 2 and sole ZnO. Importantly, the partially negative charged MoS 2 layer is beneficial to the nucleation and growth of ZnO nanosheets under the effect of electrostatic interactions. Classic methylene blue, which possesses different affinities to dsDNA and ssDNA, was adopted as the measure signal to confirm the immobilization and hybridization of DNA on ZnO nanosheets and pursue the optimal synthetic conditions. And the results demonstrated that the free-standing ZnO/MoS 2 nanosheets had low detection limit (6.6×10 -16 M) and has a positive influence on DNA immobilization and hybridization. Copyright © 2016 Elsevier B.V. All rights reserved.

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

PubMed Central

McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2005-01-01

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions. PMID:15933716

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage.

PubMed

McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

2005-07-06

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.

Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

PubMed

Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

2016-03-01

Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

Alignment of Gold Nanoparticle-Decorated DNA Origami Nanotubes: Substrate Prepatterning versus Molecular Combing.

PubMed

Teschome, Bezu; Facsko, Stefan; Gothelf, Kurt V; Keller, Adrian

2015-11-24

DNA origami has become an established technique for designing well-defined nanostructures with any desired shape and for the controlled arrangement of functional nanostructures with few nanometer resolution. These unique features make DNA origami nanostructures promising candidates for use as scaffolds in nanoelectronics and nanophotonics device fabrication. Consequently, a number of studies have shown the precise organization of metallic nanoparticles on various DNA origami shapes. In this work, we fabricated large arrays of aligned DNA origami decorated with a high density of gold nanoparticles (AuNPs). To this end, we first demonstrate the high-yield assembly of high-density AuNP arrangements on DNA origami adsorbed to Si surfaces with few unbound background nanoparticles by carefully controlling the concentrations of MgCl2 and AuNPs in the hybridization buffer and the hybridization time. Then, we evaluate two methods, i.e., hybridization to prealigned DNA origami and molecular combing in a receding meniscus, with respect to their potential to yield large arrays of aligned AuNP-decorated DNA origami nanotubes. Because of the comparatively low MgCl2 concentration required for the efficient immobilization of the AuNPs, the prealigned DNA origami become mobile and displaced from their original positions, thereby decreasing the alignment yield. This increased mobility, on the other hand, makes the adsorbed origami susceptible to molecular combing, and a total alignment yield of 86% is obtained in this way.

Structure of a DNA glycosylase that unhooks interstrand cross-links

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

Mullins, Elwood A.; Warren, Garrett M.; Bradley, Noah P.

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protectsmore » its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.« less

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2010-01-01

Summary The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease. PMID:20948980

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-01-01

The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease.

Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

NASA Astrophysics Data System (ADS)

Suzuki, Yuki; Endo, Masayuki; Sugiyama, Hiroshi

2015-08-01

Self-assembly is a ubiquitous approach to the design and fabrication of novel supermolecular architectures. Here we report a strategy termed `lipid-bilayer-assisted self-assembly' that is used to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported zwitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and self-assembled into large micrometre-sized lattices in their lateral dimensions. Using high-speed atomic force microscopy imaging, a variety of dynamic processes involved in the formation of the lattice, such as fusion, reorganization and defect filling, are successfully visualized. The surface modifiability of the assembled lattice is also demonstrated by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffolds for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space.

Lipid-bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures

PubMed Central

Endo, Masayuki; Sugiyama, Hiroshi

2015-01-01

Self-assembly is a ubiquitous approach to the design and fabrication of novel supermolecular architectures. Here we report a strategy termed ‘lipid-bilayer-assisted self-assembly' that is used to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported zwitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and self-assembled into large micrometre-sized lattices in their lateral dimensions. Using high-speed atomic force microscopy imaging, a variety of dynamic processes involved in the formation of the lattice, such as fusion, reorganization and defect filling, are successfully visualized. The surface modifiability of the assembled lattice is also demonstrated by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffolds for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space. PMID:26310995

Kinetics of DNA-mediated docking reactions between vesicles tethered to supported lipid bilayers

PubMed Central

Chan, Yee-Hung M.; Lenz, Peter; Boxer, Steven G.

2007-01-01

Membrane–membrane recognition and binding are crucial in many biological processes. We report an approach to studying the dynamics of such reactions by using DNA-tethered vesicles as a general scaffold for displaying membrane components. This system was used to characterize the docking reaction between two populations of tethered vesicles that display complementary DNA. Deposition of vesicles onto a supported lipid bilayer was performed by using a microfluidic device to prevent mixing of the vesicles in bulk during sample preparation. Once tethered onto the surface, vesicles mixed via two-dimensional diffusion. DNA-mediated docking of two reacting vesicles results in their colocalization after collision and their subsequent tandem motion. Individual docking events and population kinetics were observed via epifluorescence microscopy. A lattice-diffusion simulation was implemented to extract from experimental data the probability, Pdock, that a collision leads to docking. For individual vesicles displaying small numbers of docking DNA, Pdock shows a first-order relationship with copy number as well as a strong dependence on the DNA sequence. Both trends are explained by a model that includes both tethered vesicle diffusion on the supported bilayer and docking DNA diffusion over each vesicle's surface. These results provide the basis for the application of tethered vesicles to study other membrane reactions including protein-mediated docking and fusion. PMID:18025472

Multilayer DNA Origami Packed on a Square Lattice

PubMed Central

Ke, Yonggang; Douglas, Shawn M.; Liu, Minghui; Sharma, Jaswinder; Cheng, Anchi; Leung, Albert; Liu, Yan; Shih, William M.; Yan, Hao

2009-01-01

Molecular self-assembly using DNA as a structural building block has proven to be an efficient route to the construction of nanoscale objects and arrays of increasing complexity. Using the remarkable “scaffolded DNA origami” strategy, Rothemund demonstrated that a long single-stranded DNA from a viral genome (M13) can be folded into a variety of custom two-dimensional (2D) shapes using hundreds of short synthetic DNA molecules as staple strands. More recently, we generalized a strategy to build custom-shaped, three-dimensional (3D) objects formed as pleated layers of helices constrained to a honeycomb lattice, with precisely controlled dimensions ranging from 10 to 100 nm. Here we describe a more compact design for 3D origami, with layers of helices packed on a square lattice, that can be folded successfully into structures of designed dimensions in a one-step annealing process, despite the increased density of DNA helices. A square lattice provides a more natural framework for designing rectangular structures, the option for a more densely packed architecture, and the ability to create surfaces that are more flat than is possible with the honeycomb lattice. Thus enabling the design and construction of custom 3D shapes from helices packed on a square lattice provides a general foundational advance for increasing the versatility and scope of DNA nanotechnology. PMID:19807088

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro.

PubMed

Sil, Payel; Yoo, Dae-Goon; Floyd, Madison; Gingerich, Aaron; Rada, Balazs

2016-06-18

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

PubMed Central

Almeida, Karen H.; Sobol, Robert W.

2007-01-01

Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

Tissue-engineered trachea regeneration using decellularized trachea matrix treated with laser micropore technique.

PubMed

Xu, Yong; Li, Dan; Yin, Zongqi; He, Aijuan; Lin, Miaomiao; Jiang, Gening; Song, Xiao; Hu, Xuefei; Liu, Yi; Wang, Jinpeng; Wang, Xiaoyun; Duan, Liang; Zhou, Guangdong

2017-08-01

Tissue-engineered trachea provides a promising approach for reconstruction of long segmental tracheal defects. However, a lack of ideal biodegradable scaffolds greatly restricts its clinical translation. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration owing to natural tubular structure, cartilage matrix components, and biodegradability. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. To address these problems, a laser micropore technique (LMT) was applied in the current study to modify trachea sample porosity to facilitate decellular treatment and cell ingrowth. Decellularization processing demonstrated that cells in LMT treated samples were more easily removed compared with untreated native trachea. Furthermore, after optimizing the protocols of LMT and decellular treatments, the LMT-treated DTM (LDTM) could retain their original tubular shape with only mild extracellular matrix damage. After seeding with chondrocytes and culture in vitro for 8 weeks, the cell-LDTM constructs formed tubular cartilage with relatively homogenous cell distribution in both micropores and bilateral surfaces. In vivo results further confirmed that the constructs could form mature tubular cartilage with increased DNA and cartilage matrix contents, as well as enhanced mechanical strength, compared with native trachea. Collectively, these results indicate that LDTM is an ideal scaffold for tubular cartilage regeneration and, thus, provides a promising strategy for functional reconstruction of trachea cartilage. Lacking ideal biodegradable scaffolds greatly restricts development of tissue-engineered trachea. Decellularized trachea matrix (DTM) is considered a proper scaffold for trachea cartilage regeneration. However, cell residual and low porosity of DTM easily result in immunogenicity and incomplete cartilage regeneration. By laser micropore technique (LMT), the current study efficiently enhanced the porosity and decellularized efficacy of DTM. The LMT-treated DTM basically retained the original tubular shape with mild matrix damage. After chondrocyte seeding followed by in vitro culture and in vivo implantation, the constructs formed mature tubular cartilage with matrix content and mechanical strength similar to native trachea. The current study provides an ideal scaffold and a promising strategy for cartilage regeneration and functional reconstruction of trachea. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

PubMed

Sail, Vibhavari; Rizzo, Alessandro A; Chatterjee, Nimrat; Dash, Radha C; Ozen, Zuleyha; Walker, Graham C; Korzhnev, Dmitry M; Hadden, M Kyle

2017-07-21

Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

Exploring the Interactions of the Dietary Plant Flavonoids Fisetin and Naringenin with G-Quadruplex and Duplex DNA, Showing Contrasting Binding Behavior: Spectroscopic and Molecular Modeling Approaches.

PubMed

Bhattacharjee, Snehasish; Chakraborty, Sandipan; Sengupta, Pradeep K; Bhowmik, Sudipta

2016-09-01

Guanine-rich sequences have the propensity to fold into a four-stranded DNA structure known as a G-quadruplex (G4). G4 forming sequences are abundant in the promoter region of several oncogenes and become a key target for anticancer drug binding. Here we have studied the interactions of two structurally similar dietary plant flavonoids fisetin and naringenin with G4 as well as double stranded (duplex) DNA by using different spectroscopic and modeling techniques. Our study demonstrates the differential binding ability of the two flavonoids with G4 and duplex DNA. Fisetin more strongly interacts with parallel G4 structure than duplex DNA, whereas naringenin shows stronger binding affinity to duplex rather than G4 DNA. Molecular docking results also corroborate our spectroscopic results, and it was found that both of the ligands are stacked externally in the G4 DNA structure. C-ring planarity of the flavonoid structure appears to be a crucial factor for preferential G4 DNA recognition of flavonoids. The goal of this study is to explore the critical effects of small differences in the structure of closely similar chemical classes of such small molecules (flavonoids) which lead to the contrasting binding properties with the two different forms of DNA. The resulting insights may be expected to facilitate the designing of the highly selective G4 DNA binders based on flavonoid scaffolds.

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

Stella, Stefano; University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen; Molina, Rafael

Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-bindingmore » domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.« less

DNA origami applications in cancer therapy.

PubMed

Udomprasert, Anuttara; Kangsamaksin, Thaned

2017-08-01

Due to the complexity and heterogeneity of cancer, the development of cancer diagnosis and therapy is still progressing, and a complete understanding of cancer biology remains elusive. Recently, cancer nanomedicine has gained much interest as a promising diagnostic and therapeutic strategy, as a wide range of nanomaterials possess unique physical properties that can render drug delivery systems safer and more effective. Also, targeted drug delivery and precision medicine have now become a new paradigm in cancer therapy. With nanocarriers, chemotherapeutic drugs could be directly delivered into target cancer cells, resulting in enhanced efficiency with fewer side-effects. DNA, a biomolecule with molecular self-assembly properties, has emerged as a versatile nanomaterial to construct multifunctional platforms; DNA nanostructures can be modified with functional groups to improve their utilities as biosensors or drug carriers. Such applications have become possible with the advent of the scaffolded DNA origami method. This breakthrough technique in structural DNA nanotechnology provides an easier and faster way to construct DNA nanostructures with various shapes. Several experiments proved that DNA origami nanostructures possess abilities to enhance efficacies of chemotherapy, reduce adverse side-effects, and even circumvent drug resistance. Here, we highlight the principles of the DNA origami technique and its applications in cancer therapeutics and discuss current challenges and opportunities to improve cancer detection and targeted drug delivery. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

PubMed

Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

2015-08-01

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Platelets and Plasma Stimulate Sheep Rotator Cuff Tendon Tenocytes When Cultured in an Extracellular Matrix Scaffold

PubMed Central

Kelly, Brian A.; Proffen, Benedikt L.; Haslauer, Carla M.; Murray, Martha M.

2015-01-01

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: 1) Plasma (PPP), 2) Plasma and platelets (PAP), 3) Plasma and macrophages (PPPM), 4) Plasma, platelets and macrophages (PAPM), 5) Phosphate buffered saline (PBS), and 6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine if these changes in cellular function will translate into improved tendon healing. PMID:26419602

Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application.

PubMed

Urbani, Luca; Maghsoudlou, Panagiotis; Milan, Anna; Menikou, Maria; Hagen, Charlotte Klara; Totonelli, Giorgia; Camilli, Carlotta; Eaton, Simon; Burns, Alan; Olivo, Alessandro; De Coppi, Paolo

2017-01-01

Oesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for short- and long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergent-enzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes.

Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

PubMed

Kelly, Brian A; Proffen, Benedikt L; Haslauer, Carla M; Murray, Martha M

2016-04-01

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds: A combined experimental and computational approach.

PubMed

Vetsch, Jolanda Rita; Betts, Duncan Colin; Müller, Ralph; Hofmann, Sandra

2017-01-01

Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

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

Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. In this paper, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165-induced proliferation of human umbilical vein endothelialmore » cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Finally, such reagents would be useful for diagnostic and therapeutic applications.« less

Fibrochondrocyte Growth and Functionality on TiO2 Nanothin Films

PubMed Central

Ronald, Sharon; Mills, David K.

2016-01-01

Disorders affecting the temporomandibular joint (TMJ) are a long-standing health concern. TMJ disorders (TMJD) are often associated with an internal disc derangement accompanied by a suite of symptoms including joint noises, jaw dysfunction, and severe pain. The severity of patient symptoms and their reoccurrence can be alleviated to some extent with conservative therapy; however, refractory cases often require surgery that has shown only limited success. Bioengineered scaffolds with cell supportive surfaces an d nanoarchitectures that mimic TMJ tissue structure may offer an alternative treatment modality. In this study, titanium dioxide (TiO2) nanothin films, fabricated by layer-by-layer assembly, were examined as means for creating such a scaffold. The viability and growth of TMJ discal fibrochondrocytes (FCs) were assessed through MTT and DNA assays and total protein content over a 14-day experimental period. ELISA was also used to measure expression of types I and II collagen, decorin and aggrecan. Quantitative analyses demonstrated that FCs synthesized characteristic discal matrix proteins, with an increased production of type I collagen and decorin as opposed to collagen type II and aggrecan. A stimulatory effect on discal FC proliferation and extracellular matrix (ECM) expression with thicker nanofilms was also observed. The cumulative results suggest that TiO2 nanofilms may have potential as a TMJ scaffolding material. PMID:27314395

Fibrochondrocyte Growth and Functionality on TiO₂ Nanothin Films.

PubMed

Ronald, Sharon; Mills, David K

2016-06-14

Disorders affecting the temporomandibular joint (TMJ) are a long-standing health concern. TMJ disorders (TMJD) are often associated with an internal disc derangement accompanied by a suite of symptoms including joint noises, jaw dysfunction, and severe pain. The severity of patient symptoms and their reoccurrence can be alleviated to some extent with conservative therapy; however, refractory cases often require surgery that has shown only limited success. Bioengineered scaffolds with cell supportive surfaces an d nanoarchitectures that mimic TMJ tissue structure may offer an alternative treatment modality. In this study, titanium dioxide (TiO₂) nanothin films, fabricated by layer-by-layer assembly, were examined as means for creating such a scaffold. The viability and growth of TMJ discal fibrochondrocytes (FCs) were assessed through MTT and DNA assays and total protein content over a 14-day experimental period. ELISA was also used to measure expression of types I and II collagen, decorin and aggrecan. Quantitative analyses demonstrated that FCs synthesized characteristic discal matrix proteins, with an increased production of type I collagen and decorin as opposed to collagen type II and aggrecan. A stimulatory effect on discal FC proliferation and extracellular matrix (ECM) expression with thicker nanofilms was also observed. The cumulative results suggest that TiO₂ nanofilms may have potential as a TMJ scaffolding material.

Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection.

PubMed

Tipper, Donald J; Szomolanyi-Tsuda, Eva

2016-01-01

Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes β-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by >98%.

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

DOE PAGES

Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.; ...

2015-03-30

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. In this paper, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165-induced proliferation of human umbilical vein endothelialmore » cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Finally, such reagents would be useful for diagnostic and therapeutic applications.« less

Fe-S Cluster Hsp70 Chaperones: The ATPase Cycle and Protein Interactions.

PubMed

Dutkiewicz, Rafal; Nowak, Malgorzata; Craig, Elizabeth A; Marszalek, Jaroslaw

2017-01-01

Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of cellular structures, and disassembly of protein complexes. The biogenesis of iron-sulfur (Fe-S) clusters is one of the critical cellular processes that require Hsp70/J-protein action. Fe-S clusters are ubiquitous cofactors critical for activity of proteins performing diverse functions in, for example, metabolism, RNA/DNA transactions, and environmental sensing. This biogenesis process can be divided into two sequential steps: first, the assembly of an Fe-S cluster on a conserved scaffold protein, and second, the transfer of the cluster from the scaffold to a recipient protein. The second step involves Hsp70/J-protein chaperones. Via binding to the scaffold, chaperones enable cluster transfer to recipient proteins. In eukaryotic cells mitochondria have a key role in Fe-S cluster biogenesis. In this review, we focus on methods that enabled us to dissect protein interactions critical for the function of Hsp70/J-protein chaperones in the mitochondrial process of Fe-S cluster biogenesis in the yeast Saccharomyces cerevisiae. © 2017 Elsevier Inc. All rights reserved.

(Poly)cation-induced protection of conventional and wireframe DNA origami nanostructures.

PubMed

Ahmadi, Yasaman; De Llano, Elisa; Barišić, Ivan

2018-04-26

DNA nanostructures hold immense potential to be used for biological and medical applications. However, they are extremely vulnerable towards salt depletion and nucleases, which are common under physiological conditions. In this contribution, we used chitosan and linear polyethyleneimine for coating and long-term stabilization of several three-dimensional DNA origami nanostructures. The impact of the degree of polymerization and the charge density of the polymer together with the N/P charge ratio (ratio of the amines in polycations to the phosphates in DNA) on the stability of encapsulated DNA origami nanostructures in the presence of nucleases and in low-salt media was examined. The polycation shells were compatible with enzyme- and aptamer-based functionalization of the DNA nanostructures. Additionally, we showed that despite being highly vulnerable to salt depletion and nucleolytic digestion, self-assembled DNA nanostructures are stable in cell culture media up to a week. This was contrary to unassembled DNA scaffolds that degraded in one hour, showing that placing DNA strands into a spatially designed configuration crucially affect the structural integrity. The stability of naked DNA nanostructures in cell culture was shown to be mediated by growth media. DNA origami nanostructures kept in growth media were significantly more resistant towards low-salt denaturation, DNase I and serum-mediated digestion than when in a conventional buffer. Moreover, we confirmed that DNA origami nanostructures remain not only structurally intact but also fully functional after exposure to cell media. Agarose gel electrophoresis and negative stain transmission electron microscopy analysis revealed the hybridization of DNA origami nanostructures to their targets in the presence of serum proteins and nucleases. The structural integrity and functionality of DNA nanostructures in physiological fluids validate their use particularly for short-time biological applications in which the shape and structural details of DNA nanodevices are functionally critical.

Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone

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

Aoyagi-Scharber, Mika, E-mail: maoyagi@bmrn.com; Gardberg, Anna S.; Yip, Bryan K.

2014-08-29

BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity. Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2), which are involved in DNA damage response, are targets of anticancer therapeutics. BMN 673 is a novel PARP1/2 inhibitor with substantially increased PARP-mediated tumor cytotoxicity and is now in later-stage clinical development for BRCA-deficient breast cancers.more » In co-crystal structures, BMN 673 is anchored to the nicotinamide-binding pocket via an extensive network of hydrogen-bonding and π-stacking interactions, including those mediated by active-site water molecules. The novel di-branched scaffold of BMN 673 extends the binding interactions towards the outer edges of the pocket, which exhibit the least sequence homology among PARP enzymes. The crystallographic structural analyses reported here therefore not only provide critical insights into the molecular basis for the exceptionally high potency of the clinical development candidate BMN 673, but also new opportunities for increasing inhibitor selectivity.« less

Development and characterization of decellularized human nasoseptal cartilage matrix for use in tissue engineering.

PubMed

Graham, M Elise; Gratzer, Paul F; Bezuhly, Michael; Hong, Paul

2016-10-01

Reconstruction of cartilage defects in the head and neck can require harvesting of autologous cartilage grafts, which can be associated with donor site morbidity. To overcome this limitation, tissue-engineering approaches may be used to generate cartilage grafts. The objective of this study was to decellularize and characterize human nasoseptal cartilage with the aim of generating a biological scaffold for cartilage tissue engineering. Laboratory study using nasoseptal cartilage. Remnant human nasoseptal cartilage specimens were collected and subjected to a novel decellularization treatment. The decellularization process involved several cycles of enzymatic detergent treatments. For characterization, decellularized and fresh (control) specimens underwent histological, biochemical, and mechanical analyses. Scanning electron microscopy and biocompatibility assay were also performed. The decellularization process had minimal effect on glycosaminoglycan content of the cartilage extracellular matrix. Deoxyribonucleic acid (DNA) analysis revealed the near-complete removal of genomic DNA from decellularized tissues. The effectiveness of the decellularization process was also confirmed on histological and scanning electron microscopic analyses. Mechanical testing results showed that the structural integrity of the decellularized tissue was maintained, and biocompatibility was confirmed. Overall, the current decellularization treatment resulted in significant reduction of genetic/cellular material with preservation of the underlying extracellular matrix structure. This decellularized material may serve as a potential scaffold for cartilage tissue engineering. N/A. Laryngoscope, 126:2226-2231, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

The Genome Sequence of a Widespread Apex Predator, the Golden Eagle (Aquila chrysaetos)

PubMed Central

Doyle, Jacqueline M.; Katzner, Todd E.; Bloom, Peter H.; Ji, Yanzhu; Wijayawardena, Bhagya K.; DeWoody, J. Andrew

2014-01-01

Biologists routinely use molecular markers to identify conservation units, to quantify genetic connectivity, to estimate population sizes, and to identify targets of selection. Many imperiled eagle populations require such efforts and would benefit from enhanced genomic resources. We sequenced, assembled, and annotated the first eagle genome using DNA from a male golden eagle (Aquila chrysaetos) captured in western North America. We constructed genomic libraries that were sequenced using Illumina technology and assembled the high-quality data to a depth of ∼40x coverage. The genome assembly includes 2,552 scaffolds >10 Kb and 415 scaffolds >1.2 Mb. We annotated 16,571 genes that are involved in myriad biological processes, including such disparate traits as beak formation and color vision. We also identified repetitive regions spanning 92 Mb (∼6% of the assembly), including LINES, SINES, LTR-RTs and DNA transposons. The mitochondrial genome encompasses 17,332 bp and is ∼91% identical to the Mountain Hawk-Eagle (Nisaetus nipalensis). Finally, the data reveal that several anonymous microsatellites commonly used for population studies are embedded within protein-coding genes and thus may not have evolved in a neutral fashion. Because the genome sequence includes ∼800,000 novel polymorphisms, markers can now be chosen based on their proximity to functional genes involved in migration, carnivory, and other biological processes. PMID:24759626

Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis.

PubMed

Princz, Lissa N; Wild, Philipp; Bittmann, Julia; Aguado, F Javier; Blanco, Miguel G; Matos, Joao; Pfander, Boris

2017-03-01

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution. © 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Continuing differentiation of human mesenchymal stem cells and induced chondrogenic and osteogenic lineages in electrospun PLGA nanofiber scaffold

PubMed Central

Xin, Xuejun; Hussain, Mohammad; Mao, Jeremy J.

2010-01-01

Nanofibers have recently gained substantial interest for potential applications in tissue engineering. The objective of this study was to determine whether electrospun nanofibers accommodate the viability, growth, and differentiation of human mesenchymal stem cells (hMSCs) as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives. Poly(D,L-lactide-co-glycolide) (PLGA) beads with a PLA:PGA ratio of 85:15 were electrospun into non-woven fibers with an average diameter of 760±210 nm. The average Young’s modulus of electrospun PLGA nanofibers was 42±26 kPa, per nanoindentation with atomic force microscopy (AFM). Human MSCs were seeded 1–4 weeks at a density of 2×106 cells/mL in PLGA nanofiber sheets. After 2 week culture on PLGA nanofiber scaffold, hMSCs remained as precursors upon immunoblotting with hKL12 antibody. SEM taken up to 7 days after cell seeding revealed that hMSCs, hMSC-Ob and hMSC-Ch apparently attached to PLGA nanofibers. The overwhelming majority of hMSCs was viable and proliferating in PLGA nanofiber scaffolds up to the tested 14 days, as assayed live/dead tests, DNA assay and BrdU. In a separate experiment, hMSCs seeded in PLGA nanofiber scaffolds were differentiated into chodrogenic and osteogenic cells. Histological assays revealed that hMSCs continuously differentiated into chondrogenic cells and osteogenic cells after 2 week incubation in PLGA nanofibers. Taken together, these data represent an original investigation of continuous differentiation of hMSCs into chondrogenic and osteogenic cells in PLGA nanofiber scaffold. Consistent with previous work, these findings also suggest that nanofibers may serve as accommodative milieu for not only hMSCs, but also as a 3D carrier vehicle for lineage specific cells. PMID:17010425

Extended Duration of Transgene Expression from Pegylated POD Nanoparticles Enables Attenuation of Photoreceptor Degeneration

PubMed Central

Binder, Christina; Cashman, Siobhan M.; Kumar-Singh, Rajendra

2013-01-01

Retinitis pigmentosa (RP) is the most genetically heterogeneous disorder known to cause blindness, involving over 50 different genes. Previously, we have described nanoparticles (NPs) 150 nm in size, comprised of a 3.5 kD peptide (POD) complexed to PEG and DNA (PEGPOD DNA). These NPs expressing GDNF enabled rescue of photoreceptor degeneration in mice up to 11 days post injection. In the current study we examine use of scaffold/ matrix attachment regions (S/MARs), CpG depletion and titration of DNA content of PEGPOD DNA NPs to extend the duration of transgene expression. S/MARs and CpGs did not significantly influence the duration of transgene expression, but did influence its stability. These parameters enabled us to extend transgene expression from 48 hours to 10 weeks. At 77 days post injection, we observed a 76% rescue of the thickness of the retinal outer nuclear layer (ONL) and at 37 days post injection we observed 53% and 55% rescue of the A and B wave ERG amplitudes respectively and 60% rescue of the ONL. Our studies suggest that PEGPOD DNA NPs have potential as gene delivery vectors for the retina. PMID:24278479

Rev1 Recruits Ung to Switch Regions and Enhances dU Glycosylation for Immunoglobulin Class Switch DNA Recombination

PubMed Central

Zan, Hong; White, Clayton A.; Thomas, Lisa M.; Mai, Thach; Li, Guideng; Xu, Zhenming; Zhang, Jinsong; Casali, Paolo

2012-01-01

SUMMARY By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response. It is initiated by AID-mediated dC deamination, yielding dUs, and dU glycosylation by Ung in antibody switch (S) region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1–/– Ung+/+ B cells reduced Ung recruitment to S regions, DNA-dU glycosylation and CSR. This together with an S region spectrum of mutations similar to that of Rev1+/+ Ung–/– B cells suggested that Rev1 operated in the same pathway as Ung, as emphasized by further decreased CSR in Rev1–/– Msh2–/– B cells. Rescue of CSR in Rev1–/– B cells by a catalytically inactive Rev1 mutant showed that the important role of Rev1 in CSR is mediated by Rev1 scaffold, not enzymatic function. PMID:23140944

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites.

PubMed

Balani, Valério Américo; de Lima Neto, Quirino Alves; Takeda, Karen Izumi; Gimenes, Fabrícia; Fiorini, Adriana; Debatisse, Michelle; Fernandez, Maria Aparecida

2010-11-01

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB.

States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM.

PubMed

Serwer, Philip; Wright, Elena T; Demeler, Borries; Jiang, Wen

2018-04-01

Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.

Synthesis and functionalization of gold nanorods for probing plasmonic enhancement mechanisms in organic photovoltaic active layers

NASA Astrophysics Data System (ADS)

Wadams, Robert Christopher

DNA nanotechnology is one of the most flourishing interdisciplinary research fields. Through the features of programmability and predictability, DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales, and more importantly, they can be used as scaffolds for organizing other nanoparticles, proteins and chemical groups. By leveraging these molecules, DNA nanostructures can be used to direct the organization of complex bio-inspired materials that may serve as smart drug delivery systems and in vitro or in vivo bio-molecular computing and diagnostic devices. In this dissertation I describe a systematic study of the thermodynamic properties of complex DNA nanostructures, including 2D and 3D DNA origami, in order to understand their assembly, stability and functionality and inform future design endeavors. It is conceivable that a more thorough understanding of DNA self-assembly can be used to guide the structural design process and optimize the conditions for assembly, manipulation, and functionalization, thus benefiting both upstream design and downstream applications. As a biocompatible nanoscale motif, the successful integration, stabilization and separation of DNA nanostructures from cells/cell lysate suggests its potential to serve as a diagnostic platform at the cellular level. Here, DNA origami was used to capture and identify multiple T cell receptor mRNA species from single cells within a mixed cell population. This demonstrates the potential of DNA nanostructure as an ideal nano scale tool for biological applications.

Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

PubMed

Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

2016-07-15

Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Micromechanical study of protein-DNA interactions and chromosomes

NASA Astrophysics Data System (ADS)

Marko, John

I will discuss micromechanics experiments that our group has used to analyze protein-DNA interactions and chromosome organization. In single-DNA experiments we have found that a feature of protein-DNA complexes is that their dissociation rates can depend strikingly on bulk solution concentrations of other proteins and DNA segments; I will describe experiments which demonstrate this effect, which can involve tens-fold changes in off-rates with submicromolar changes in solution concentrations. Second, I will discuss experiments aimed at analyzing large-scale human chromosome structure; we isolate metaphase chromosomes, which in their native form behave as remarkably elastic networks of chromatin. Exposure to DNA-cutting restriction enzymes completely eliminates this elasticity, indicating that there is not a mechanically contiguous protein ''scaffold'' from which the chromosome gains its stability. I will show results of siRNA experiments indicating that depletion of condensin proteins leads to destabilization of chromosome mechanics, indicating condensin's role as the major chromatin ''cross-linker'' in metaphase chromosomes. Finally I will discuss similar experiments on human G1 nuclei, where we use genetic and chemical modifications to separate the contributions of the nuclear lamina and chromatin to the mechanical stiffness of the nucleus as a whole. Supported by the NSF (DMR-1206868, MCB-1022117) and the NIH (GM105847, CA193419).

Temperature-Dependent Charge Transport through Individually Contacted DNA Origami-Based Au Nanowires.

PubMed

Teschome, Bezu; Facsko, Stefan; Schönherr, Tommy; Kerbusch, Jochen; Keller, Adrian; Erbe, Artur

2016-10-11

DNA origami nanostructures have been used extensively as scaffolds for numerous applications such as for organizing both organic and inorganic nanomaterials, studying single molecule reactions, and fabricating photonic devices. Yet, little has been done toward the integration of DNA origami nanostructures into nanoelectronic devices. Among other challenges, the technical difficulties in producing well-defined electrical contacts between macroscopic electrodes and individual DNA origami-based nanodevices represent a serious bottleneck that hinders the thorough characterization of such devices. Therefore, in this work, we have developed a method to electrically contact individual DNA origami-based metallic nanowires using electron beam lithography. We then characterize the charge transport of such nanowires in the temperature range from room temperature down to 4.2 K. The room temperature charge transport measurements exhibit ohmic behavior, whereas at lower temperatures, multiple charge transport mechanisms such as tunneling and thermally assisted transport start to dominate. Our results confirm that charge transport along metallized DNA origami nanostructures may deviate from pure metallic behavior due to several factors including partial metallization, seed inhomogeneities, impurities, and weak electronic coupling among AuNPs. Besides, this study further elucidates the importance of variable temperature measurements for determining the dominant charge transport mechanisms for conductive nanostructures made by self-assembly approaches.

Triplex-forming oligonucleotides: a third strand for DNA nanotechnology

PubMed Central

2018-01-01

Abstract DNA self-assembly has proved to be a useful bottom-up strategy for the construction of user-defined nanoscale objects, lattices and devices. The design of these structures has largely relied on exploiting simple base pairing rules and the formation of double-helical domains as secondary structural elements. However, other helical forms involving specific non-canonical base-base interactions have introduced a novel paradigm into the process of engineering with DNA. The most notable of these is a three-stranded complex generated by the binding of a third strand within the duplex major groove, generating a triple-helical (‘triplex’) structure. The sequence, structural and assembly requirements that differentiate triplexes from their duplex counterparts has allowed the design of nanostructures for both dynamic and/or structural purposes, as well as a means to target non-nucleic acid components to precise locations within a nanostructure scaffold. Here, we review the properties of triplexes that have proved useful in the engineering of DNA nanostructures, with an emphasis on applications that hitherto have not been possible by duplex formation alone. PMID:29228337

A "turn-on" fluorescent copper biosensor based on DNA cleavage-dependent graphene-quenched DNAzyme.

PubMed

Liu, Meng; Zhao, Huimin; Chen, Shuo; Yu, Hongtao; Zhang, Yaobin; Quan, Xie

2011-06-15

A novel and promising "turn-on" fluorescent Cu(2+) biosensor is designed based on graphene-DNAzyme catalytic beacon. Due to the essential surface and quenching properties of two-dimensional graphene, it can function as both "scaffold" and "quencher" of the Cu(2+)-dependent DNAzyme, facilitating the formation of self-assembled graphene-quenched DNAzyme complex. However, Cu(2+)-induced catalytic reaction disturbs the graphene-DNAzyme conformation, which will produce internal DNA cleavage-dependent effect. In this case, the quenched fluorescence in graphene-DNAzyme is quickly recovered to a large extent in 15 min. Compared with common DNAzyme-based sensors, the presented graphene-based catalytic beacon greatly improves the signal-to-background ratio, hence increasing the sensitivity (LOD=0.365 nM). Furthermore, the controllable DNA cleavage reaction provides an original and alternative internal method to regulate the interaction between graphene and DNA relative to the previous external sequence-specific hybridization-dependent regulation, which will open new opportunities for nucleic studies and sensing applications in the future. Copyright © 2011 Elsevier B.V. All rights reserved.

Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

PubMed Central

Boer, D. Roeland; Ruiz-Masó, José Angel; Rueda, Manuel; Petoukhov, Maxim V.; Machón, Cristina; Svergun, Dmitri I.; Orozco, Modesto; del Solar, Gloria; Coll, Miquel

2016-01-01

DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation. PMID:26875695

Peering down the barrel of a bacteriophage portal: the genome packaging and release valve in p22.

PubMed

Tang, Jinghua; Lander, Gabriel C; Olia, Adam S; Olia, Adam; Li, Rui; Casjens, Sherwood; Prevelige, Peter; Cingolani, Gino; Baker, Timothy S; Johnson, John E

2011-04-13

The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 Å resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by Olia et al. (2011) were fitted into this reconstruction. The interpreted density map revealed that the 150 Å, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Components of Adenovirus Genome Packaging

PubMed Central

Ahi, Yadvinder S.; Mittal, Suresh K.

2016-01-01

Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

DNA-Assembled Advanced Plasmonic Architectures.

PubMed

Liu, Na; Liedl, Tim

2018-03-28

The interaction between light and matter can be controlled efficiently by structuring materials at a length scale shorter than the wavelength of interest. With the goal to build optical devices that operate at the nanoscale, plasmonics has established itself as a discipline, where near-field effects of electromagnetic waves created in the vicinity of metallic surfaces can give rise to a variety of novel phenomena and fascinating applications. As research on plasmonics has emerged from the optics and solid-state communities, most laboratories employ top-down lithography to implement their nanophotonic designs. In this review, we discuss the recent, successful efforts of employing self-assembled DNA nanostructures as scaffolds for creating advanced plasmonic architectures. DNA self-assembly exploits the base-pairing specificity of nucleic acid sequences and allows for the nanometer-precise organization of organic molecules but also for the arrangement of inorganic particles in space. Bottom-up self-assembly thus bypasses many of the limitations of conventional fabrication methods. As a consequence, powerful tools such as DNA origami have pushed the boundaries of nanophotonics and new ways of thinking about plasmonic designs are on the rise.

Novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-ones act as covalent poisons of human topoisomerase IIα.

PubMed

Infante Lara, Lorena; Sledge, Alexis; Laradji, Amine; Okoro, Cosmas O; Osheroff, Neil

2017-02-01

A number of topoisomerase II-targeted anticancer drugs, including amsacrine, utilize an acridine or related aromatic core as a scaffold. Therefore, to further explore the potential of acridine-related compounds to act as topoisomerase II poisons, we synthesized a series of novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives and examined their ability to enhance DNA cleavage mediated by human topoisomerase IIα. Derivatives containing a H, Cl, F, and Br at C7 enhanced enzyme-mediated double-stranded DNA cleavage ∼5.5- to 8.5-fold over baseline, but were less potent than amsacrine. The inclusion of an amino group at C9 was critical for activity. The compounds lost their activity against topoisomerase IIα in the presence of a reducing agent, displayed no activity against the catalytic core of topoisomerase IIα, and inhibited DNA cleavage when incubated with the enzyme prior to the addition of DNA. These findings strongly suggest that the compounds act as covalent, rather than interfacial, topoisomerase II poisons. Published by Elsevier Ltd.

A structural-alphabet-based strategy for finding structural motifs across protein families

PubMed Central

Wu, Chih Yuan; Chen, Yao Chi; Lim, Carmay

2010-01-01

Proteins with insignificant sequence and overall structure similarity may still share locally conserved contiguous structural segments; i.e. structural/3D motifs. Most methods for finding 3D motifs require a known motif to search for other similar structures or functionally/structurally crucial residues. Here, without requiring a query motif or essential residues, a fully automated method for discovering 3D motifs of various sizes across protein families with different folds based on a 16-letter structural alphabet is presented. It was applied to structurally non-redundant proteins bound to DNA, RNA, obligate/non-obligate proteins as well as free DNA-binding proteins (DBPs) and proteins with known structures but unknown function. Its usefulness was illustrated by analyzing the 3D motifs found in DBPs. A non-specific motif was found with a ‘corner’ architecture that confers a stable scaffold and enables diverse interactions, making it suitable for binding not only DNA but also RNA and proteins. Furthermore, DNA-specific motifs present ‘only’ in DBPs were discovered. The motifs found can provide useful guidelines in detecting binding sites and computational protein redesign. PMID:20525797

Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2010-01-01

Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures.

PubMed

Koussa, Mounir A; Sotomayor, Marcos; Wong, Wesley P

2014-05-15

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes. Copyright © 2014. Published by Elsevier Inc.

Protocol for sortase-mediated construction of DNA-protein hybrids and functional nanostructures

PubMed Central

Koussa, Mounir A.; Sotomayor, Marcos; Wong, Wesley P.

2014-01-01

Recent methods in DNA nanotechnology are enabling the creation of intricate nanostructures through the use of programmable, bottom-up self-assembly. However, structures consisting only of DNA are limited in their ability to act on other biomolecules. Proteins, on the other hand, perform a variety of functions on biological materials, but directed control of the self-assembly process remains a challenge. While DNA-protein hybrids have the potential to provide the best-of-both-worlds, they can be difficult to create as many of the conventional techniques for linking proteins to DNA render proteins dysfunctional. We present here a sortase-based protocol for covalently coupling proteins to DNA with minimal disturbance to protein function. To accomplish this we have developed a two-step process. First, a small synthetic peptide is bioorthogonally and covalently coupled to a DNA oligo using click chemistry. Next, the DNA-peptide chimera is covalently linked to a protein of interest under protein-compatible conditions using the enzyme sortase. Our protocol allows for the simple coupling and purification of a functional DNA-protein hybrid. We use this technique to form oligos bearing cadherin-23 and protocadherin-15 protein fragments. Upon incorporation into a linear M13 scaffold, these protein-DNA hybrids serve as the gate to a binary nanoswitch. The outlined protocol is reliable and modular, facilitating the construction of libraries of oligos and proteins that can be combined to form functional DNA-protein nanostructures. These structures will enable a new class of functional nanostructures, which could be used for therapeutic and industrial processes. PMID:24568941

Carbazole ligands as c-myc G-quadruplex binders.

PubMed

Głuszyńska, Agata; Juskowiak, Bernard; Kuta-Siejkowska, Martyna; Hoffmann, Marcin; Haider, Shozeb

2018-07-15

The interactions of c-myc G-quadruplex with three carbazole derivatives were investigated by UV-Vis spectrophotometry, fluorescence, CD spectroscopy, and molecular modeling. The results showed that a combination of carbazole scaffold functionalized with ethyl, triazole and imidazole groups resulted in stabilization of the intramolecular G-quadruplex formed by the DNA sequence derived from the NHE III 1 region of c-myc oncogene (Pu22). Binding to the G-quadruplex Pu22 resulted in the significant increase in fluorescence intensity of complexed ligands 1-3. All ligands were capable of interacting with G4 DNA with binding stoichiometry indicating that two ligand molecules bind to G-quadruplex with comparable affinity, which agrees with binding model of end-stacking on terminal G-tetrads. Copyright © 2018 Elsevier B.V. All rights reserved.

Mass effect of redox reactions: A novel mode for surface plasmon resonance-based bioanalysis.

PubMed

Yuan, Pei-Xin; Deng, Sheng-Yuan; Xin, Peng; Ji, Xu-Bo; Shan, Dan; Cosnier, Serge

2015-12-15

The pursuit of more specific and sensitive response is a perpetual goal for modern bioassays. This work proposed a novel label-free strategy about redox-related mass effect based on the surface plasmon resonance (SPR) technique for ultrasensitive determination of DNA. The protocol starts with the modification of SPR gilded disk with the capture DNA (cDNA). After the conjugation of immobilized cDNA with the target DNA (tDNA), the hybridization chain reaction was triggered by the introduction of mutual partial complementary primers to elongate the terminal into a nanoscale duplex. As it is reported that porphyrin could intercalate into the grooves of the double-stranded DNA (dsDNA) scaffold, multiple positive-charged Fe(III)meso-tetra(N-methyl-4-pyridyl) porphine (FeTMPyP) with symmetric structure were uptaken for in situ formation of porphyrin-dsDNA complex. Given FeTMPyP a highly efficient catalysis for the peroxide reduction, its presence as a biomimetic cofactor was validated via circular dichroism and UV-vis spectroscopy, demonstrating a tight binding as well as high catalytic activity and stability. Using 4-chloro-1-naphthol as a proton donor, the catalytic reduction of H2O2 would oxidize it into insoluble benzo-4-chloro-hexadienone, which simultaneously deposited on the heterogeneous interface, leading to a significant amplification in both SPR response and topological height profile. The signal increment was proportional to the concentration of tDNA, thus an ultrasensitive SPR-based DNA assay was developed with a linear range over four orders of magnitudes and a sub-femtomolar detection limit of 0.73 fM. The developed methodology exemplifies a different way of thinking about mass-sensing modes, extending conventional SPR-based DNA analysis to relevant biomedical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Improvement of the Threespine Stickleback Genome Using a Hi-C-Based Proximity-Guided Assembly.

PubMed

Peichel, Catherine L; Sullivan, Shawn T; Liachko, Ivan; White, Michael A

2017-09-01

Scaffolding genomes into complete chromosome assemblies remains challenging even with the rapidly increasing sequence coverage generated by current next-generation sequence technologies. Even with scaffolding information, many genome assemblies remain incomplete. The genome of the threespine stickleback (Gasterosteus aculeatus), a fish model system in evolutionary genetics and genomics, is not completely assembled despite scaffolding with high-density linkage maps. Here, we first test the ability of a Hi-C based proximity-guided assembly (PGA) to perform a de novo genome assembly from relatively short contigs. Using Hi-C based PGA, we generated complete chromosome assemblies from a distribution of short contigs (20-100 kb). We found that 96.40% of contigs were correctly assigned to linkage groups (LGs), with ordering nearly identical to the previous genome assembly. Using available bacterial artificial chromosome (BAC) end sequences, we provide evidence that some of the few discrepancies between the Hi-C assembly and the existing assembly are due to structural variation between the populations used for the 2 assemblies or errors in the existing assembly. This Hi-C assembly also allowed us to improve the existing assembly, assigning over 60% (13.35 Mb) of the previously unassigned (~21.7 Mb) contigs to LGs. Together, our results highlight the potential of the Hi-C based PGA method to be used in combination with short read data to perform relatively inexpensive de novo genome assemblies. This approach will be particularly useful in organisms in which it is difficult to perform linkage mapping or to obtain high molecular weight DNA required for other scaffolding methods. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A novel member of the SAF (scaffold attachment factor)-box protein family inhibits gene expression and induces apoptosis

PubMed Central

Chan, Ching Wan; Lee, Youn-Bok; Uney, James; Flynn, Andrea; Tobias, Jonathan H.; Norman, Michael

2007-01-01

The SLTM [SAF (scaffold attachment factor)-like transcription modulator] protein contains a SAF-box DNA-binding motif and an RNA-binding domain, and shares an overall identity of 34% with SAFB1 {scaffold attachment factor-B1; also known as SAF-B (scaffold attachment factor B), HET [heat-shock protein 27 ERE (oestrogen response element) and TATA-box-binding protein] or HAP (heterogeneous nuclear ribonucleoprotein A1-interacting protein)}. Here, we show that SLTM is localized to the cell nucleus, but excluded from nucleoli, and to a large extent it co-localizes with SAFB1. In the nucleus, SLTM has a punctate distribution and it does not co-localize with SR (serine/arginine) proteins. Overexpression of SAFB1 has been shown to exert a number of inhibitory effects, including suppression of oestrogen signalling. Although SLTM also suppressed the ability of oestrogen to activate a reporter gene in MCF-7 breast-cancer cells, inhibition of a constitutively active β-galactosidase gene suggested that this was primarily the consequence of a generalized inhibitory effect on transcription. Measurement of RNA synthesis, which showed a particularly marked inhibition of [3H]uridine incorporation into mRNA, supported this conclusion. In addition, analysis of cell-cycle parameters, chromatin condensation and cytochrome c release showed that SLTM induced apoptosis in a range of cultured cell lines. Thus the inhibitory effects of SLTM on gene expression appear to result from generalized down-regulation of mRNA synthesis and initiation of apoptosis consequent upon overexpressing the protein. While indicating a crucial role for SLTM in cellular function, these results also emphasize the need for caution when interpreting phenotypic changes associated with manipulation of protein expression levels. PMID:17630952

Generation of a Close-to-Native In Vitro System to Study Lung Cells-Extracellular Matrix Crosstalk.

PubMed

Garlíková, Zuzana; Silva, Ana Catarina; Rabata, Anas; Potěšil, David; Ihnatová, Ivana; Dumková, Jana; Koledová, Zuzana; Zdráhal, Zbyněk; Vinarský, Vladimír; Hampl, Aleš; Pinto-do-Ó, Perpétua; Nascimento, Diana Santos

2018-01-01

Extracellular matrix (ECM) is an essential component of the tissue microenvironment, actively shaping cellular behavior. In vitro culture systems are often poor in ECM constituents, thus not allowing for naturally occurring cell-ECM interactions. This study reports on a straightforward and efficient method for the generation of ECM scaffolds from lung tissue and its subsequent in vitro application using primary lung cells. Mouse lung tissue was subjected to decellularization with 0.2% sodium dodecyl sulfate, hypotonic solutions, and DNase. Resultant ECM scaffolds were devoid of cells and DNA, whereas lung ECM architecture of alveolar region and blood and airway networks were preserved. Scaffolds were predominantly composed of core ECM and ECM-associated proteins such as collagens I-IV, nephronectin, heparan sulfate proteoglycan core protein, and lysyl oxidase homolog 1, among others. When homogenized and applied as coating substrate, ECM supported the attachment of lung fibroblasts (LFs) in a dose-dependent manner. After ECM characterization and biocompatibility tests, a novel in vitro platform for three-dimensional (3D) matrix repopulation that permits live imaging of cell-ECM interactions was established. Using this system, LFs colonized the ECM scaffolds, displaying a close-to-native morphology in intimate interaction with the ECM fibers, and showed nuclear translocation of the mechanosensor yes-associated protein (YAP), when compared with cells cultured in two dimensions. In conclusion, we developed a 3D-like culture system, by combining an efficient decellularization method with a live-imaging culture platform, to replicate in vitro native lung cell-ECM crosstalk. This is a valuable system that can be easily applied to other organs for ECM-related drug screening, disease modeling, and basic mechanistic studies.

Assembling programmable FRET-based photonic networks using designer DNA scaffolds

PubMed Central

Buckhout-White, Susan; Spillmann, Christopher M; Algar, W. Russ; Khachatrian, Ani; Melinger, Joseph S.; Goldman, Ellen R.; Ancona, Mario G.; Medintz, Igor L.

2014-01-01

DNA demonstrates a remarkable capacity for creating designer nanostructures and devices. A growing number of these structures utilize Förster resonance energy transfer (FRET) as part of the device's functionality, readout or characterization, and, as device sophistication increases so do the concomitant FRET requirements. Here we create multi-dye FRET cascades and assess how well DNA can marshal organic dyes into nanoantennae that focus excitonic energy. We evaluate 36 increasingly complex designs including linear, bifurcated, Holliday junction, 8-arm star and dendrimers involving up to five different dyes engaging in four-consecutive FRET steps, while systematically varying fluorophore spacing by Förster distance (R0). Decreasing R0 while augmenting cross-sectional collection area with multiple donors significantly increases terminal exciton delivery efficiency within dendrimers compared with the first linear constructs. Förster modelling confirms that best results are obtained when there are multiple interacting FRET pathways rather than independent channels by which excitons travel from initial donor(s) to final acceptor. PMID:25504073

Enzyme-linked DNA dendrimer nanosensors for acetylcholine

PubMed Central

Walsh, Ryan; Morales, Jennifer M.; Skipwith, Christopher G.; Ruckh, Timothy T.; Clark, Heather A.

2015-01-01

It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience. PMID:26442999

Enzyme-linked DNA dendrimer nanosensors for acetylcholine.

PubMed

Walsh, Ryan; Morales, Jennifer M; Skipwith, Christopher G; Ruckh, Timothy T; Clark, Heather A

2015-10-07

It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience.

Enzyme-linked DNA dendrimer nanosensors for acetylcholine

NASA Astrophysics Data System (ADS)

Walsh, Ryan; Morales, Jennifer M.; Skipwith, Christopher G.; Ruckh, Timothy T.; Clark, Heather A.

2015-10-01

It is currently difficult to measure small dynamics of molecules in the brain with high spatial and temporal resolution while connecting them to the bigger picture of brain function. A step towards understanding the underlying neural networks of the brain is the ability to sense discrete changes of acetylcholine within a synapse. Here we show an efficient method for generating acetylcholine-detecting nanosensors based on DNA dendrimer scaffolds that incorporate butyrylcholinesterase and fluorescein in a nanoscale arrangement. These nanosensors are selective for acetylcholine and reversibly respond to levels of acetylcholine in the neurophysiological range. This DNA dendrimer architecture has the potential to overcome current obstacles to sensing in the synaptic environment, including the nanoscale size constraints of the synapse and the ability to quantify the spatio-temporal fluctuations of neurotransmitter release. By combining the control of nanosensor architecture with the strategic placement of fluorescent reporters and enzymes, this novel nanosensor platform can facilitate the development of new selective imaging tools for neuroscience.

Kinetics and Mechanism of Mammalian Mitochondrial Ribosome Assembly.

PubMed

Bogenhagen, Daniel F; Ostermeyer-Fay, Anne G; Haley, John D; Garcia-Diaz, Miguel

2018-02-13

Mammalian mtDNA encodes only 13 proteins, all essential components of respiratory complexes, synthesized by mitochondrial ribosomes. Mitoribosomes contain greatly truncated RNAs transcribed from mtDNA, including a structural tRNA in place of 5S RNA as a scaffold for binding 82 nucleus-encoded proteins, mitoribosomal proteins (MRPs). Cryoelectron microscopy (cryo-EM) studies have determined the structure of the mitoribosome, but its mechanism of assembly is unknown. Our SILAC pulse-labeling experiments determine the rates of mitochondrial import of MRPs and their assembly into intact mitoribosomes, providing a basis for distinguishing MRPs that bind at early and late stages in mitoribosome assembly to generate a working model for mitoribosome assembly. Mitoribosome assembly is a slow process initiated at the mtDNA nucleoid driven by excess synthesis of individual MRPs. MRPs that are tightly associated in the structure frequently join the complex in a coordinated manner. Clinically significant MRP mutations reported to date affect proteins that bind early on during assembly. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure.

PubMed

Suzuki, Yuki; Endo, Masayuki; Cañas, Cristina; Ayora, Silvia; Alonso, Juan C; Sugiyama, Hiroshi; Takeyasu, Kunio

2014-06-01

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells)

PubMed Central

Serwer, Philip

2011-01-01

I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells) include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1) initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2) are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke. PMID:21994778

Identification of Leishmania donovani Topoisomerase 1 inhibitors via intuitive scaffold hopping and bioisosteric modification of known Top 1 inhibitors

NASA Astrophysics Data System (ADS)

Mamidala, Rajinikanth; Majumdar, Papiya; Jha, Kunal Kumar; Bathula, Chandramohan; Agarwal, Rahul; Chary, M. Thirumala; Mazumdar, H. K.; Munshi, Parthapratim; Sen, Subhabrata

2016-05-01

A library of arylidenefuropyridinediones was discovered as potent inhibitors of Leishmania donovani Topoisomerase 1 (LdTop1) where the active molecules displayed considerable inhibition with single digit micromolar EC50 values. This molecular library was designed via intuitive scaffold hopping and bioisosteric modification of known topoisomerase 1 inhibitors such as camptothecin, edotecarin and etc. The design was rationalized by molecular docking analysis of the compound prototype with human topoisomerase 1 (HTop1) and Leishmania donovani topoisomerase 1(LdTop1). The most active compound 4 displayed no cytotoxicity against normal mammalian COS7 cell line (~100 fold less inhibition at the EC50). Similar to camptothecin, 4 interacted with free LdTop1 as observed in the preincubation DNA relaxation inhibition experiment. It also displayed anti-protozoal activity against Leishmania donovani promastigote. Crystal structure investigation of 4 and its molecular modelling with LdTop1 revealed putative binding sites in the enzyme that could be harnessed to generate molecules with better potency.

The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold.

PubMed

Knott, Gavin J; Bond, Charles S; Fox, Archa H

2016-05-19

Nuclear proteins are often given a concise title that captures their function, such as 'transcription factor,' 'polymerase' or 'nuclear-receptor.' However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein-protein and protein-nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold

PubMed Central

Knott, Gavin J.; Bond, Charles S.; Fox, Archa H.

2016-01-01

Nuclear proteins are often given a concise title that captures their function, such as ‘transcription factor,’ ‘polymerase’ or ‘nuclear-receptor.’ However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein–protein and protein–nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology. PMID:27084935

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

PubMed

Sugitani, Norie; Voehler, Markus W; Roh, Michelle S; Topolska-Woś, Agnieszka M; Chazin, Walter J

2017-10-13

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Determination of the melon chloroplast and mitochondrial genome sequences reveals that the largest reported mitochondrial genome in plants contains a significant amount of DNA having a nuclear origin

PubMed Central

2011-01-01

Background The melon belongs to the Cucurbitaceae family, whose economic importance among vegetable crops is second only to Solanaceae. The melon has a small genome size (454 Mb), which makes it suitable for molecular and genetic studies. Despite similar nuclear and chloroplast genome sizes, cucurbits show great variation when their mitochondrial genomes are compared. The melon possesses the largest plant mitochondrial genome, as much as eight times larger than that of other cucurbits. Results The nucleotide sequences of the melon chloroplast and mitochondrial genomes were determined. The chloroplast genome (156,017 bp) included 132 genes, with 98 single-copy genes dispersed between the small (SSC) and large (LSC) single-copy regions and 17 duplicated genes in the inverted repeat regions (IRa and IRb). A comparison of the cucumber and melon chloroplast genomes showed differences in only approximately 5% of nucleotides, mainly due to short indels and SNPs. Additionally, 2.74 Mb of mitochondrial sequence, accounting for 95% of the estimated mitochondrial genome size, were assembled into five scaffolds and four additional unscaffolded contigs. An 84% of the mitochondrial genome is contained in a single scaffold. The gene-coding region accounted for 1.7% (45,926 bp) of the total sequence, including 51 protein-coding genes, 4 conserved ORFs, 3 rRNA genes and 24 tRNA genes. Despite the differences observed in the mitochondrial genome sizes of cucurbit species, Citrullus lanatus (379 kb), Cucurbita pepo (983 kb) and Cucumis melo (2,740 kb) share 120 kb of sequence, including the predicted protein-coding regions. Nevertheless, melon contained a high number of repetitive sequences and a high content of DNA of nuclear origin, which represented 42% and 47% of the total sequence, respectively. Conclusions Whereas the size and gene organisation of chloroplast genomes are similar among the cucurbit species, mitochondrial genomes show a wide variety of sizes, with a non-conserved structure both in gene number and organisation, as well as in the features of the noncoding DNA. The transfer of nuclear DNA to the melon mitochondrial genome and the high proportion of repetitive DNA appear to explain the size of the largest mitochondrial genome reported so far. PMID:21854637

Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η*

PubMed Central

Su, Yan; Egli, Martin; Guengerich, F. Peter

2016-01-01

Ribonucleotides and 2′-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2′-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2′-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2′-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 103-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2′-deoxyguanosine with a significant propeller twist. As a result, the 2′-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion. PMID:26740629

Xenogeneic Acellular Conjunctiva Matrix as a Scaffold of Tissue-Engineered Corneal Epithelium

PubMed Central

Zhao, Haifeng; Qu, Mingli; Wang, Yao; Wang, Zhenyu; Shi, Weiyun

2014-01-01

Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface. PMID:25375996

Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

PubMed

Raisin, Sophie; Belamie, Emmanuel; Morille, Marie

2016-10-01

Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sequence analysis of the genome of carnation (Dianthus caryophyllus L.).

PubMed

Yagi, Masafumi; Kosugi, Shunichi; Hirakawa, Hideki; Ohmiya, Akemi; Tanase, Koji; Harada, Taro; Kishimoto, Kyutaro; Nakayama, Masayoshi; Ichimura, Kazuo; Onozaki, Takashi; Yamaguchi, Hiroyasu; Sasaki, Nobuhiro; Miyahara, Taira; Nishizaki, Yuzo; Ozeki, Yoshihiro; Nakamura, Noriko; Suzuki, Takamasa; Tanaka, Yoshikazu; Sato, Shusei; Shirasawa, Kenta; Isobe, Sachiko; Miyamura, Yoshinori; Watanabe, Akiko; Nakayama, Shinobu; Kishida, Yoshie; Kohara, Mitsuyo; Tabata, Satoshi

2014-06-01

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. 'Francesco' was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568,887,315 bp, consisting of 45,088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16,644 bp and 60,737 bp, respectively, and the longest scaffold was 1,287,144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼ 98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp. © The Author 2013. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors.

PubMed

Miyazaki, Tatsuya; Miyauchi, Satoshi; Matsuzaka, Satoshi; Yamagishi, Chie; Kobayashi, Kohei

2010-05-01

Tissue-engineered cartilage may be expected to serve as an alternative to autologous chondrocyte transplantation treatment. Several methods for producing cartilaginous tissue have been reported. In this study, we describe the production of scaffold-free stiff cartilaginous tissue of pig and human, using allogeneic serum and growth factors. The tissue was formed in a mold using chondrocytes recovered from alginate bead culture and maintained in a medium with transforming growth factor-beta and several other additives. In the case of porcine tissue, the tear strength of the tissue and the contents of proteoglycan (PG) and collagen per unit of DNA increased dose-dependently with transforming growth factor-beta. The length of culture was significantly and positively correlated with thickness, tear strength, and PG and collagen contents. Tear strength showed positive high correlations with both PG and collagen contents. A positive correlation was also seen between PG content and collagen content. Similar results were obtained with human cartilaginous tissue formed from chondrocytes expanded in monolayer culture. Further, an in vivo pilot study using pig articular cartilage defect model demonstrated that the cartilaginous tissue was well integrated with surrounding tissue at 13 weeks after the implantation. In conclusion, we successfully produced implantable scaffold-free stiff cartilaginous tissue, which characterized high PG and collagen contents.

Enhancement of tissue engineered bone formation by a low pressure system improving cell seeding and medium perfusion into a porous scaffold.

PubMed

Wang, Juyong; Asou, Yoshinori; Sekiya, Ichiro; Sotome, Shinichi; Orii, Hisaya; Shinomiya, Kenichi

2006-05-01

To obtain more extensive bone formation in composites of porous ceramics and bone marrow stromal cells (BMSCs), we hypothesized that a low-pressure system would serve to facilitate the perfusion of larger number of BMSCs into the porous scaffold, enhancing bone formation within the composites. After culturing BMSCs in osteogenic medium, porous blocks of beta-tricalcium phosphate (beta-TCP) were soaked in the cell suspension. Composites of the block and BMSCs were put immediately into a vacuum desiccator. Low pressure was applied to the low pressure group, while controls were left at atmospheric pressure. Composites were incubated in vitro or subcutaneously implanted into syngeneic rats, then analyzed biologically and histologically. In the in vitro group, cell suspension volume, cell seeding efficiency, alkaline phosphatase (ALP) activity, and DNA content in the beta-TCP blocks were significantly higher in low pressure group than in the controls. Scanning electron microscopy (SEM) demonstrated that a greater number of cells covered the central parts of the composites in the low pressure group. ALP activity in the composites was increased at 3 and 6 weeks after implantation into rats. Histomorphometric analysis revealed more uniform and extensive bone formation in the low pressure group than in the controls. The application of low pressure during the seeding of BMSCs in perfusing medium into a porous scaffold is useful for tissue-engineered bone formation.

Sodium hydroxide based non-detergent decellularizing solution for rat lung.

PubMed

Sengyoku, Hideyori; Tsuchiya, Tomoshi; Obata, Tomohiro; Doi, Ryoichiro; Hashimoto, Yasumasa; Ishii, Mitsutoshi; Sakai, Hiromi; Matsuo, Naoto; Taniguchi, Daisuke; Suematsu, Takashi; Lawn, Murray; Matsumoto, Keitaro; Miyazaki, Takuro; Nagayasu, Takeshi

2018-06-11

Lung transplantation is the last option for the treatment of end stage chronic lung disorders. Because the shortage of donor lung organs represents the main hurdle, lung regeneration has been considered to overcome this hurdle. Recellularization of decellularized organ scaffold is a promising option for organ regeneration. Although detergents are ordinarily used for decellularization, other approaches are possible. Here we used high alkaline (pH12) sodium hydroxide (NaOH)-PBS solution without detergents for lung decellularization and compared the efficacy on DNA elimination and ECM preservation with detergent based decellularization solutions CHAPS and SDS. Immunohistochemical image analysis showed that cell components were removed by NaOH solution as well as other detergents. A Collagen and GAG assay showed that the collagen reduction of the NaOH group was comparable to that of the CHAPS and SDS groups. However, DNA reduction was more significant in the NaOH group than in other groups (p < 0.0001). The recellularization of HUVEC revealed cell attachment was not inferior to that of the SDS group. Ex vivo functional analysis showed 100% oxygen ventilation increased oxygen partial pressure as artificial hemoglobin vesicle-PBS solution passed through regenerated lungs in the SDS or NaOH group. It was concluded that the NaOH-PBS based decellularization solution was comparable to ordinal decellularizaton solutions and competitive in cost effectiveness and residues in the decellularized scaffold negligible, thus providing another potential option to detergent for future clinical usage.

Sub–100-nm metafluorophores with digitally tunable optical properties self-assembled from DNA

PubMed Central

Woehrstein, Johannes B.; Strauss, Maximilian T.; Ong, Luvena L.; Wei, Bryan; Zhang, David Y.; Jungmann, Ralf; Yin, Peng

2017-01-01

Fluorescence microscopy allows specific target detection down to the level of single molecules and has become an enabling tool in biological research. To transduce the biological information to an imageable signal, we have developed a variety of fluorescent probes, such as organic dyes or fluorescent proteins with different colors. Despite their success, a limitation on constructing small fluorescent probes is the lack of a general framework to achieve precise and programmable control of critical optical properties, such as color and brightness. To address this challenge, we introduce metafluorophores, which are constructed as DNA nanostructure–based fluorescent probes with digitally tunable optical properties. Each metafluorophore is composed of multiple organic fluorophores, organized in a spatially controlled fashion in a compact sub–100-nm architecture using a DNA nanostructure scaffold. Using DNA origami with a size of 90 × 60 nm2, substantially smaller than the optical diffraction limit, we constructed small fluorescent probes with digitally tunable brightness, color, and photostability and demonstrated a palette of 124 virtual colors. Using these probes as fluorescent barcodes, we implemented an assay for multiplexed quantification of nucleic acids. Additionally, we demonstrated the triggered in situ self-assembly of fluorescent DNA nanostructures with prescribed brightness upon initial hybridization to a nucleic acid target. PMID:28691083

Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.

PubMed

Ma, Caili; Lv, Linlin; Liu, Yu; Yu, Yanni; You, Renchuan; Yang, Jicheng; Li, Mingzhong

2014-06-01

Vascularization is a crucial challenge in tissue engineering. One solution for this problem is to implant scaffolds that contain functional genes that promote vascularization by providing angiogenic growth factors via a gene delivery carrier. Poly(ethylenimine) (PEI) is a gene delivery carrier with high transfection efficiency but with cytotoxicity. To solve this problem, we utilized Antheraea pernyi silk fibroin (ASF), which has favorable cytocompatibility and biodegradability, RGD sequences and a negative charge, in conjunction with PEI, as the delivery vector for vascular endothelial growth factor (VEGF) 165-angiopoietin-1 (Ang-1) dual gene simultaneous expression plasmid, creating an ASF/PEI/pDNA complex. The results suggested that the zeta potential of the ASF/PEI/pDNA complex was significantly lower than that of the PEI/pDNA complex. Decreased nitrogen and increased oxygen on the surface of the complex demonstrated that the ASF had successfully combined with the surface of the PEI/pDNA. Furthermore, the complexes resisted digestion by nucleic acid enzymes and degradation by serum. L929 cells were cultured and transfected in vitro and improved cytotoxicity was found when the cells were transfected with ASF/PEI/pDNA compared with PEI/pDNA. In addition, the transfection efficiency and VEGF secretion increased. In general, this study provides a novel method for decreasing the cytotoxicity of PEI gene delivery vectors and increasing transfection efficiency of angiogenesis-related genes.

Chromatin tethering effects of hNopp140 are involved in the spatial organization of nucleolus and the rRNA gene transcription

PubMed Central

Tsai, Yi-Tzang; Lin, Chen-I; Chen, Hung-Kai; Lee, Kuo-Ming; Hsu, Chia-Yi; Yang, Shun-Jen

2008-01-01

The short arms of five human acrocentric chromosomes contain ribosomal gene (rDNA) clusters where numerous mini-nucleoli arise at the exit of mitosis. These small nucleoli tend to coalesce into one or a few large nucleoli during interphase by unknown mechanisms. Here, we demonstrate that the N- and C-terminal domains of a nucleolar protein, hNopp140, bound respectively to α-satellite arrays and rDNA clusters of acrocentric chromosomes for nucleolar formation. The central acidic-and-basic repeated domain of hNopp140, possessing a weak self-self interacting ability, was indispensable for hNopp140 to build up a nucleolar round-shaped structure. The N- or the C-terminally truncated hNopp140 caused nucleolar segregation and was able to alter locations of the rDNA transcription, as mediated by detaching the rDNA repeats from the acrocentric α-satellite arrays. Interestingly, an hNopp140 mutant, made by joining the N- and C-terminal domains but excluding the entire central repeated region, induced nucleolar disruption and global chromatin condensation. Furthermore, RNAi knockdown of hNopp140 resulted in dispersion of the rDNA and acrocentric α-satellite sequences away from nucleolus that was accompanied by rDNA transcriptional silence. Our findings indicate that hNopp140, a scaffold protein, is involved in the nucleolar assembly, fusion, and maintenance. PMID:18253863

Highly fluorescent peptide nanoribbon impregnated with Sn-porphyrin as a potent DNA sensor.

PubMed

Parayil, Sreenivasan Koliyat; Lee, Jooran; Yoon, Minjoong

2013-05-01

Highly fluorescent and thermo-stable peptide nanoribbons (PNRs) were fabricated by solvothermal self-assembly of a single peptide (D,D-diphenyl alanine peptides) with Sn-porphyrin (trans-dihydroxo[5,10,15,20-tetrakis(p-tolyl)porphyrinato] Sn(IV) (SnTTP(OH)2)). The structural characterization of the as-prepared nanoribbons was performed by transmitting electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM), FT-IR and Raman spectroscopy, indicating that the lipophilic Sn-porphyrins are impregnated into the porous surface formed in the process of nanoribbon formation through intermolecular hydrogen bonding of the peptide main chains. Consequently the Sn-porphyrin-impregnated peptide nanoribbons (Sn-porphyrin-PNRs) exhibited typical UV-visible absorption spectrum of the monomer porphyrin with a red shifted Q-band, and their fluorescence quantum yield was observed to be enhanced compared to that of free Sn-porphyrin. Interestingly the fluorescence intensity and lifetimes of Sn-porphyrin-PNRs were selectively affected upon interaction with nucleotide base sequences of DNA while those of free Sn-porphyrins were not affected by binding with any of the DNA studied, indicating that DNA-induced changes in the fluorescence properties of Sn-porphyrin-PNRs are due to interaction between DNA and the PNR scaffold. These results imply that Sn-porphyrin-PNR will be useful as a potent fluorescent protein analogue and as a biocompatible DNA sensor.

Colorimetric monitoring of nanometer distance changes in DNA-templated plasmon rulers (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lermusiaux, Laurent; Bidault, Sebastien

2016-03-01

The nanometer-scale sensitivity of plasmon coupling allows the translation of minute morphological changes in nanostructures into macroscopic optical signals. In particular, single nanostructure scattering spectroscopy provides a direct estimation of interparticle distances in gold nanoparticle (AuNP) dimers linked by a short DNA double-strand [M. P. Busson et al, Nano Lett. 11, 5060 (2011)]. We demonstrate here that this spectroscopic information can be inferred from simple widefield measurements on a calibrated color camera [L. Lermusiaux et al, ACS Nano 9, 978 (2015)]. This allows us to analyze the influence of electrostatic and steric interparticle interactions on the morphology of DNA-templated AuNP groupings. Furthermore, polarization-resolved measurements on a color CCD provide a parallel imaging of AuNP dimer orientations. We apply this spectroscopic characterization to identify dimers featuring two different conformations of the same DNA template. In practice, the biomolecular scaffold contains a hairpin-loop that opens after hybridization to a specific DNA sequence and increases the interparticle distance [L. Lermusiaux et al, ACS Nano 6, 10992 (2012)]. These results open exciting perspectives for the parallel sensing of single specific DNA strands using plasmon rulers. We discuss the limits of this approach in terms of the physicochemical stability and reactivity of these nanostructures and demonstrate the importance of engineering the AuNP surface chemistry, in particular using amphiphilic ligands [L. Lermusiaux and S. Bidault, Small (2015), in press].

A new biochromatography model based on DNA origami assembled PPARγ: construction and evaluation.

PubMed

Zhou, Jie; Meng, Lingchang; Sun, Chong; Chen, Shanshan; Sun, Fang; Luo, Pei; Zhao, Yongxing

2017-05-01

As drug targets, receptors have potential to screen drugs. Silica is an attractive support to immobilize receptors; however, the lack of biocompatibility makes it easier for receptors to lose bioactivity, which remains an obstacle to its widespread use. With the advantage of biocompatibility, DNA origami can be used as a biological carrier to improve the biocompatibility of silica and assemble receptors. In this study, a new biochromatography model based on DNA origami was constructed. A large quantity of M13ssDNA was used as a scaffold, leading to significant costs, so M13ssDNA was self-produced from the bacteriophage particles. This approach is demonstrated using the ligand binding domain of gamma isoform peroxisome proliferator-activated receptor (PPARγ-LBD) as a research object. PPARγ-LBD was assembled on DNA origami carrier and then coupled on the surface of silica. The products were packed into the column as stationary phase to construct the biochromatography with the ability to recognize drugs. Affinity and specificity of the biochromatography model were evaluated by HPLC. The final results showed that the biochromatography could recognize rosiglitazone specifically, which further proved that the model could screen chemical compositions interacted with PPARγ. It was the first time to take advantage of DNA origami to assemble PPARγ to construct biochromatography. The new biochromatography model has the advantages of being efficient, convenient, and high-throughput. This method affords a new way to rapidly and conveniently screen active ingredients from complex sample plant extracts and natural product-like libraries.

Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration

PubMed Central

Sato, Ayako; Kajiya, Hiroshi; Mori, Nana; Sato, Hironobu; Fukushima, Tadao; Kido, Hirofumi

2017-01-01

The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration. PMID:28060874

Stabilising the Herpes Simplex Virus capsid by DNA packaging

NASA Astrophysics Data System (ADS)

Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

2009-03-01

Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

Human autoantibody to topoisomerase II.

PubMed

Hoffmann, A; Heck, M M; Bordwell, B J; Rothfield, N F; Earnshaw, W C

1989-02-01

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.

Human umbilical cord derived matrix: A scaffold suitable for tissue engineering application.

PubMed

Dan, Pan; Velot, Émilie; Mesure, Benjamin; Groshenry, Guillaume; Bacharouche, Jalal; Decot, Véronique; Menu, Patrick

2017-01-01

Human tissue derived natural extracellular matrix (ECM) has great potential in tissue engineering. We sought to isolate extracellular matrix derived from human umbilical cord and test its potential in tissue engineering. An enzymatic method was applied to isolate and solubilized complete human umbilical cord derived matrix (hUCM). The obtained solution was analyzed for growth factors, collagen and residual DNA contents, then used to coat 2D and 3D surfaces for cell culture application. The hUCM was successfully isolated with trypsin digestion to acquire a solution containing various growth factors and collagen but no residual DNA. This hUCM solution can form a coating on 2D and 3D substrates suitable cell culture. We developed a new matrix derived from human source that can be further used in tissue engineering.

Cascade DNA nanomachine and exponential amplification biosensing.

PubMed

Xu, Jianguo; Wu, Zai-Sheng; Shen, Weiyu; Xu, Huo; Li, Hongling; Jia, Lee

2015-11-15

DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques.

PubMed

van 't Hoff, Marcel; Reuter, Marcel; Dryden, David T F; Oheim, Martin

2009-09-21

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

Hydrodynamic compression of young and adult rat osteoblast-like cells on titanium fiber mesh.

PubMed

Walboomers, X F; Elder, S E; Bumgardner, J D; Jansen, J A

2006-01-01

Living bone cells are responsive to mechanical loading. Consequently, numerous in vitro models have been developed to examine the application of loading to cells. However, not all systems are suitable for the fibrous and porous three-dimensional materials, which are preferable for tissue repair purposes, or for the production of tissue engineering scaffolds. For three-dimensional applications, mechanical loading of cells with either fluid flow systems or hydrodynamic pressure systems has to be considered. Here, we aimed to evaluate the response of osteoblast-like cells to hydrodynamic compression, while growing in a three-dimensional titanium fiber mesh scaffolding material. For this purpose, a custom hydrodynamic compression chamber was built. Bone marrow cells were obtained from the femora of young (12-day-old) or old (1-year-old) rats, and precultured in the presence of dexamethasone and beta-glycerophosphate to achieve an osteoblast-like phenotype. Subsequently, cells were seeded onto the titanium mesh scaffolds, and subjected to hydrodynamic pressure, alternating between 0.3 to 5.0 MPa at 1 Hz, at 15-min intervals for a total of 60 min per day for up to 3 days. After pressurization, cell viability was checked. Afterward, DNA levels, alkaline phosphatase (ALP) activity, and extracellular calcium content were measured. Finally, all specimens were observed with scanning electron microscopy. Cell viability studies showed that the applied pressure was not harmful to the cells. Furthermore, we found that cells were able to detect the compression forces, because we did see evident effects on the cell numbers of the cells derived from old animals. However, there were no other changes in the cells under pressure. Finally, it was also noticeable that cells from old animals did not express ALP activity, but did show similar calcified extracellular matrix formation to the cells from young animals. In conclusion, the difference in DNA levels as reaction toward pressure, and the difference in ALP levels, suggest that the osteogenic properties of bone marrow-derived osteoblast-like cells are different with respect to the age of the donor. (c) 2005 Wiley Periodicals, Inc

Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds: A combined experimental and computational approach

PubMed Central

Betts, Duncan Colin; Müller, Ralph; Hofmann, Sandra

2017-01-01

Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading. PMID:28686698

Synthesis and biological evaluation of novel N-phenyl ureidobenzenesulfonate derivatives as potential anticancer agents. Part 2. Modulation of the ring B.

PubMed

Gagné-Boulet, Mathieu; Moussa, Hanane; Lacroix, Jacques; Côté, Marie-France; Masson, Jean-Yves; Fortin, Sébastien

2015-10-20

DNA double strand-breaks (DSBs) are the most deleterious lesions that can affect the genome of living beings and are lethal if not quickly and properly repaired. Recently, we discovered a new family of anticancer agents designated as N-phenyl ureidobenzenesulfonates (PUB-SOs) that are blocking the cells cycle progression in S-phase and inducing DNA DSBs. Previously, we have studied the effect of several modifications on the molecular scaffold of PUB-SOs on their cytocidal properties. However, the effect of the nature and the position of substituents on the aromatic ring B is still poorly studied. In this study, we report the preparation and the biological evaluation of 45 new PUB-SO derivatives substituted by alkyl, alkoxy, halogen and nitro groups at different positions on the aromatic ring B. All PUB-SOs were active in the submicromolar to low micromolar range (0.24-20 μM). The cell cycle progression analysis showed that PUB-SOs substituted at position 2 by alkyl, halogen or nitro groups or substituted at position 4 by a hydroxyl group arrest the cell cycle progression in S-phase. Interestingly, all others PUB-SOs substituted at positions 3 and 4 arrested the cell cycle in G2/M-phase. PUB-SOs arresting the cell cycle progression in S-phase also induced the phosphorylation of H2AX (γH2AX) which is indicating the generation of DNA DSBs. We evidenced that few modifications on the ring B of PUB-SOs scaffold lead to cytocidal derivatives arresting the cell cycle in S-phase and inducing γH2AX and DSBs. In addition, this study shows that these new anticancer agents are promising and could be used as alternative to circumvent some of the biopharmaceutical complications that might be encountered during the development of PUB-SOs. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

PubMed Central

Liao, Sumei; Klein, Marlise I.; Heim, Kyle P.; Fan, Yuwei; Bitoun, Jacob P.; Ahn, San-Joon; Burne, Robert A.; Koo, Hyun; Brady, L. Jeannine

2014-01-01

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. PMID:24748612

SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.

PubMed

Kaltenbach, Miriam; Stein, Viktor; Hollfelder, Florian

2011-09-19

Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

PubMed

Yuan, Quan; McHenry, Charles S

2009-11-13

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

Few mitochondrial DNA sequences are inserted into the turkey (Meleagris gallopavo) nuclear genome: evolutionary analyses and informativity in the domestic lineage.

PubMed

Schiavo, G; Strillacci, M G; Ribani, A; Bovo, S; Roman-Ponce, S I; Cerolini, S; Bertolini, F; Bagnato, A; Fontanesi, L

2018-06-01

Mitochondrial DNA (mtDNA) insertions have been detected in the nuclear genome of many eukaryotes. These sequences are pseudogenes originated by horizontal transfer of mtDNA fragments into the nuclear genome, producing nuclear DNA sequences of mitochondrial origin (numt). In this study we determined the frequency and distribution of mtDNA-originated pseudogenes in the turkey (Meleagris gallopavo) nuclear genome. The turkey reference genome (Turkey_2.01) was aligned with the reference linearized mtDNA sequence using last. A total of 32 numt sequences (corresponding to 18 numt regions derived by unique insertional events) were identified in the turkey nuclear genome (size ranging from 66 to 1415 bp; identity against the modern turkey mtDNA corresponding region ranging from 62% to 100%). Numts were distributed in nine chromosomes and in one scaffold. They derived from parts of 10 mtDNA protein-coding genes, ribosomal genes, the control region and 10 tRNA genes. Seven numt regions reported in the turkey genome were identified in orthologues positions in the Gallus gallus genome and therefore were present in the ancestral genome that in the Cretaceous originated the lineages of the modern crown Galliformes. Five recently integrated turkey numts were validated by PCR in 168 turkeys of six different domestic populations. None of the analysed numts were polymorphic (i.e. absence of the inserted sequence, as reported in numts of recent integration in other species), suggesting that the reticulate speciation model is not useful for explaining the origin of the domesticated turkey lineage. © 2018 Stichting International Foundation for Animal Genetics.

In vitro engineering of fibrocartilage using CDMP1 induced dermal fibroblasts and polyglycolide.

PubMed

Zhao, Guiqing; Yin, Shuo; Liu, Guangpeng; Cen, Lian; Sun, Jian; Zhou, Heng; Liu, Wei; Cui, Lei; Cao, Yilin

2009-07-01

This study was designed to explore the feasibility of using cartilage-derived morphogenetic protein-1 (CDMP1) induced dermal fibroblasts (DFs) as seed cells and polyglycolide (PGA) as scaffold for fibrocartilage engineering. DFs isolated from canine were expanded and seeded on PGA scaffold to fabricate cell/scaffold constructs which were cultured with or without CDMP1. Proliferation and differentiation of DFs in different constructs were determined by DNA assay and glycosaminoglycan (GAG) production. Histological and immunohistochemical staining of the constructs after being in vitro cultured for 4 and 6 weeks were carried out to observe the fibrocartilage formation condition. The fibrocartilage-specific gene expression by cells in the constructs was analyzed by real-time PCR. It was shown that in the presence of CDMP1 the proliferation and GAG synthesis of DFs were significantly enhanced compared to those without CDMP1. Fibrocartilage-like tissue was formed in the CDMP1 induced construct after being cultured for 4 weeks, and it became more matured at 6 weeks as stronger staining for GAG and higher gene expression of collagen type II was observed. Since only weak staining for GAG and collagen type II was observed for the construct engineered without CDMP1, the induction effect on the fibrocartilage engineering can be ascertained when using DFs as seed cells. Furthermore, the potential of using DFs as seed cells to engineer fibrocartilage is substantiated and further study on using the engineered tissue to repair fibrocartilage defects is currently ongoing in our group.

Cartilage engineering using chondrocyte cell sheets and its application in reconstruction of microtia.

PubMed

Zhou, Libin; Ding, Ruiying; Li, Baowei; Han, Haolun; Wang, Hongnan; Wang, Gang; Xu, Bingxin; Zhai, Suoqiang; Wu, Wei

2015-01-01

The imperfections of scaffold materials have hindered the clinical application of cartilage tissue engineering. The recently developed cell-sheet technique is adopted to engineer tissues without scaffold materials, thus is considered being potentially able to overcome the problems concerning the scaffold imperfections. This study constructed monolayer and bilayer chondrocyte cell sheets and harvested the sheets with cell scraper instead of temperature-responsive culture dishes. The properties of the cultured chondrocyte cell sheets and the feasibility of cartilage engineering using the chondrocyte cell sheets was further investigated via in vitro and in vivo study. Primary extracellular matrix (ECM) formation and type II collagen expression was detected in the cell sheets during in vitro culture. After implanted into nude mice for 8 weeks, mature cartilage discs were harvested. The morphology of newly formed cartilage was similar in the constructs originated from monolayer and bilayer chondrocyte cell sheet. The chondrocytes were located within evenly distributed ovoid lacunae. Robust ECM formation and intense expression of type II collagen was observed surrounding the evenly distributed chondrocytes in the neocartilages. Biochemical analysis showed that the DNA contents of the neocartilages were higher than native human costal cartilage; while the contents of the main component of ECM, glycosaminoglycan and hydroxyproline, were similar to native human costal cartilage. In conclusion, the chondrocyte cell sheet constructed using the simple and low-cost technique is basically the same with the cell sheet cultured and harvested in temperature-responsive culture dishes, and can be used for cartilage tissue engineering.

Recent development in deciphering the structure of luminescent silver nanodots

NASA Astrophysics Data System (ADS)

Choi, Sungmoon; Yu, Junhua

2017-05-01

Matrix-stabilized silver clusters and stable luminescent few-atom silver clusters, referred to as silver nanodots, show notable difference in their photophysical properties. We present recent research on deciphering the nature of silver clusters and nanodots and understanding the factors that lead to variations in luminescent mechanisms. Due to their relatively simple structure, the matrix-stabilized clusters have been well studied. However, the single-stranded DNA (ssDNA)-stabilized silver nanodots that show the most diverse emission wavelengths and the best photophysical properties remain mysterious species. It is clear that their photophysical properties highly depend on their protection scaffolds. Analyses from combinations of high-performance liquid chromatography, inductively coupled plasma-atomic emission spectroscopy, electrophoresis, and mass spectrometry indicate that about 10 to 20 silver atoms form emissive complexes with ssDNA. However, it is possible that not all of the silver atoms in the complex form effective emission centers. Investigation of the nanodot structure will help us understand why luminescent silver nanodots are stable in aqueous solution and how to further improve their chemical and photophysical properties.

Chromatin-Bound Cullin-Ring Ligases: Regulatory Roles in DNA Replication and Potential Targeting for Cancer Therapy

PubMed Central

Jang, Sang-Min; Redon, Christophe E.; Aladjem, Mirit I.

2018-01-01

Cullin-RING (Really Interesting New Gene) E3 ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases, are functional multi-subunit complexes including substrate receptors, adaptors, cullin scaffolds, and RING-box proteins. CRLs are responsible for ubiquitination of ~20% of cellular proteins and are involved in diverse biological processes including cell cycle progression, genome stability, and oncogenesis. Not surprisingly, cullins are deregulated in many diseases and instances of cancer. Recent studies have highlighted the importance of CRL-mediated ubiquitination in the regulation of DNA replication/repair, including specific roles in chromatin assembly and disassembly of the replication machinery. The development of novel therapeutics targeting the CRLs that regulate the replication machinery and chromatin in cancer is now an attractive therapeutic strategy. In this review, we summarize the structure and assembly of CRLs and outline their cellular functions and their diverse roles in cancer, emphasizing the regulatory functions of nuclear CRLs in modulating the DNA replication machinery. Finally, we discuss the current strategies for targeting CRLs against cancer in the clinic. PMID:29594129

Alginate and DNA Gels Are Suitable Delivery Systems for Diabetic Wound Healing.

PubMed

Tellechea, Ana; Silva, Eduardo A; Min, Jianghong; Leal, Ermelindo C; Auster, Michael E; Pradhan-Nabzdyk, Leena; Shih, William; Mooney, David J; Veves, Aristidis

2015-06-01

Diabetic foot ulcers (DFU) represent a severe health problem and an unmet clinical challenge. In this study, we tested the efficacy of novel biomaterials in improving wound healing in mouse models of diabetes mellitus (DM). The biomaterials are composed of alginate- and deoxyribonucleic acid (DNA)-based gels that allow incorporation of effector cells, such as outgrowth endothelial cells (OEC), and provide sustained release of bioactive factors, such as neuropeptides and growth factors, which have been previously validated in experimental models of DM wound healing or hind limb ischemia. We tested these biomaterials in mice and demonstrate that they are biocompatible and can be injected into the wound margins without major adverse effects. In addition, we show that the combination of OEC and the neuropeptide Substance P has a better healing outcome than the delivery of OEC alone, while subtherapeutic doses of vascular endothelial growth factor (VEGF) are required for the transplanted cells to exert their beneficial effects in wound healing. In summary, alginate and DNA scaffolds could serve as potential delivery systems for the next-generation DFU therapies. © The Author(s) 2015.

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

DOE PAGES

Stewart, Jaimie Marie; Subramanian, Hari K. K.; Franco, Elisa

2017-02-16

Rational design of nucleic acidmolecules yields selfassembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. We demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 m in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowlymore » annealed, and a one-pot transcription and anneal procedure. We then identify the tile nick position as a structural requirement for lattice formation. These results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components.« less

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

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

Stewart, Jaimie Marie; Subramanian, Hari K. K.; Franco, Elisa

Rational design of nucleic acidmolecules yields selfassembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. We demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 m in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowlymore » annealed, and a one-pot transcription and anneal procedure. We then identify the tile nick position as a structural requirement for lattice formation. These results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components.« less

Polymerization amplified SPR-DNA assay on noncovalently functionalized graphene.

PubMed

Yuan, Pei-Xin; Deng, Sheng-Yuan; Yao, Chuan-Guang; Wan, Ying; Cosnier, Serge; Shan, Dan

2017-03-15

A highly efficient surface plasmon resonance (SPR)-based DNA assay was developed, by employing noncovalently functionalized graphene nanosheets as a substrate, and enzymatic catalysis-induced polymerization as mass relay. The objective of this strategy was manifold: first of all, to sensitize the overall SPR output by in situ optimized electrogeneration of graphene thin-film, which was characterized by atomic force microscopic topography; secondly, to regulate the self-assembly and orientation of biotinylated capture probes on nickel-chelated nitrilotriacetic acid (NTA) scaffolds, that anchored onto graphene-supported pyrenyl derivatives; and lastly, to synergize the signal amplification via real-time conversion of the additive aniline into polyaniline precipitation by horseradish peroxidase-tagged reporters. With this setup, a precise and replicable DNA sensing platform for specific targets was achieved with a detection limit down to femtomolar, thus demonstrating a beneficial exploration and exploitation of two-dimensional nanomaterials as unique SPR infrastructure. The possibility of such ″bottom-up″ architecture mounted with ″top-down″ weight reactor would be most likely extensible and adaptable to protein determinations. Copyright © 2016 Elsevier B.V. All rights reserved.

Self-assembly of multi-stranded RNA motifs into lattices and tubular structures

PubMed Central

Stewart, Jaimie Marie; Subramanian, Hari K. K.

2017-01-01

Abstract Rational design of nucleic acid molecules yields self-assembling scaffolds with increasing complexity, size and functionality. It is an open question whether design methods tailored to build DNA nanostructures can be adapted to build RNA nanostructures with comparable features. Here we demonstrate the formation of RNA lattices and tubular assemblies from double crossover (DX) tiles, a canonical motif in DNA nanotechnology. Tubular structures can exceed 1 μm in length, suggesting that this DX motif can produce very robust lattices. Some of these tubes spontaneously form with left-handed chirality. We obtain assemblies by using two methods: a protocol where gel-extracted RNA strands are slowly annealed, and a one-pot transcription and anneal procedure. We identify the tile nick position as a structural requirement for lattice formation. Our results demonstrate that stable RNA structures can be obtained with design tools imported from DNA nanotechnology. These large assemblies could be potentially integrated with a variety of functional RNA motifs for drug or nanoparticle delivery, or for colocalization of cellular components. PMID:28204562

Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae): the first eukaryotic species description combining transcriptomic, DNA barcoding and micro-CT imaging data

PubMed Central

2013-01-01

Abstract We demonstrate how a classical taxonomic description of a new species can be enhanced by applying new generation molecular methods, and novel computing and imaging technologies. A cave-dwelling centipede, Eupolybothrus cavernicolus Komerički & Stoev sp. n. (Chilopoda: Lithobiomorpha: Lithobiidae), found in a remote karst region in Knin, Croatia, is the first eukaryotic species for which, in addition to the traditional morphological description, we provide a fully sequenced transcriptome, a DNA barcode, detailed anatomical X-ray microtomography (micro-CT) scans, and a movie of the living specimen to document important traits of its ex-situ behaviour. By employing micro-CT scanning in a new species for the first time, we create a high-resolution morphological and anatomical dataset that allows virtual reconstructions of the specimen and subsequent interactive manipulation to test the recently introduced ‘cybertype’ notion. In addition, the transcriptome was recorded with a total of 67,785 scaffolds, having an average length of 812 bp and N50 of 1,448 bp (see GigaDB). Subsequent annotation of 22,866 scaffolds was conducted by tracing homologs against current available databases, including Nr, SwissProt and COG. This pilot project illustrates a workflow of producing, storing, publishing and disseminating large data sets associated with a description of a new taxon. All data have been deposited in publicly accessible repositories, such as GigaScience GigaDB, NCBI, BOLD, Morphbank and Morphosource, and the respective open licenses used ensure their accessibility and re-usability. PMID:24723752

The Research of Acellular pancreatic bioscaffoldas a natural 3D platform In Vitro

NASA Astrophysics Data System (ADS)

Wang, Xin; Li, Zhao

2018-03-01

AIM: To investigate the biochemical and functional properties of a rat acellular pancreatic bioscaffold (APB). METHODS: Fresh pancreata were soaked and perfused. The histological structure, the extracellular matrix (ECM) composition, and the DNA content of the APBs were evaluated. After biocompatibility studies, the proliferation, apoptosis and differentiation of AR42J pancreatic acinar cells cultured on APBs were assessed. RESULTS: The pancreatic tissues became translucent after decellularization. The native macroscopic 3D architecture and the ECM ultrastructure were preserved, with large ductal structures and vascular tissue branching from the greater pancreatic artery, but there were no visible vascular endothelial cells, cellular components or cracked cellular debris. The ECM components, including collagen I, collagen IV, fibronectin, laminin and sGAG, were not decreased after decellularization of the APB (P>0.05) however, the DNA content was decreased significantly (P<0.05). The subcutaneous implantation sites showed low immunological response and low cytotoxicity around the APB. The proliferation rate was higher and the apoptosis rate was lower when AR42J cells were cultured on APB than when they were cultured in media alone, on artificial scaffold or ECM (P<0.05). The gene expression of pancreatic duodenal homeodomain containing transcription factor (PDX-1) and pancreatic exocrine transcription factor (PTF-1) and the protein expression of α-Amy, cytokeratin 7 (CK7) and fetal liver kinase-1 (Flk-1) were higher for the APB group than for the other groups (P<0.001). CONCLUSION: Our findings support the biological utility of whole pancreas APBs as biomaterial scaffolds, which provides an improved approach for regenerative medicine.

Lack of Association between NLGN3, NLGN4, SHANK2 and SHANK3 Gene Variants and Autism Spectrum Disorder in a Chinese Population

PubMed Central

Liu, Wenwen; Yang, Caohua; Liu, Yan; Wang, Hongyan; Gong, Xiaohong

2013-01-01

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication, absence or delay in language development, and stereotyped or repetitive behaviors. Genetic studies show that neurexin-neuroligin (NRXN-NLGN) pathway genes contribute susceptibility to ASD, which include cell adhesion molecules NLGN3, NLGN4 and scaffolding proteins SHANK2 and SHANK3. Neuroligin proteins play an important role in synaptic function and trans-synaptic signaling by interacting with presynaptic neurexins. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density. Sequence level mutations and structural variations in these genes have been identified in ASD cases, while few studies were performed in Chinese population. In this study, we examined the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). We also screened the regulatory region including the promoter region and 5′/3′ untranslated regions (UTR) and the entire coding region of NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. DNA copy number calculation in four genes showed no deletion or duplication in our cases. No missense mutations in NLGN4 were identified in our cohort. Association analysis of 6 common SNPs in NLGN4 did not find significant difference between ASD cases and controls. These findings showed that these genes may not be major disease genes in Chinese ASD cases. PMID:23468870

Lack of association between NLGN3, NLGN4, SHANK2 and SHANK3 gene variants and autism spectrum disorder in a Chinese population.

PubMed

Liu, Yanyan; Du, Yasong; Liu, Wenwen; Yang, Caohua; Liu, Yan; Wang, Hongyan; Gong, Xiaohong

2013-01-01

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication, absence or delay in language development, and stereotyped or repetitive behaviors. Genetic studies show that neurexin-neuroligin (NRXN-NLGN) pathway genes contribute susceptibility to ASD, which include cell adhesion molecules NLGN3, NLGN4 and scaffolding proteins SHANK2 and SHANK3. Neuroligin proteins play an important role in synaptic function and trans-synaptic signaling by interacting with presynaptic neurexins. Shank proteins are scaffolding molecules of excitatory synapses, which function as central organizers of the postsynaptic density. Sequence level mutations and structural variations in these genes have been identified in ASD cases, while few studies were performed in Chinese population. In this study, we examined the copy numbers of four genes NLGN4, NLGN3, SHANK2, and SHANK3 in 285 ASD cases using multiplex fluorescence competitive polymerase chain reaction (PCR). We also screened the regulatory region including the promoter region and 5'/3' untranslated regions (UTR) and the entire coding region of NLGN4 in a cohort of 285 ASD patients and 384 controls by direct sequencing of genomic DNA using the Sanger method. DNA copy number calculation in four genes showed no deletion or duplication in our cases. No missense mutations in NLGN4 were identified in our cohort. Association analysis of 6 common SNPs in NLGN4 did not find significant difference between ASD cases and controls. These findings showed that these genes may not be major disease genes in Chinese ASD cases.

Modulating the DNA affinity of Elk-1 with computationally selected mutations.

PubMed

Park, Sheldon; Boder, Eric T; Saven, Jeffery G

2005-04-22

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

Bioengineering natural product biosynthetic pathways for therapeutic applications.

PubMed

Wu, Ming-Cheng; Law, Brian; Wilkinson, Barrie; Micklefield, Jason

2012-12-01

With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Scaffold Protein TANK/I-TRAF Inhibits NF-κB Activation by Recruiting Polo-like Kinase 1

PubMed Central

Zhang, Wanqiao; Zhang, Ying; Yuan, Yanzhi; Guan, Wei; Jin, Chaozhi; Chen, Hui; Wang, Xiaohui

2010-01-01

TANK/I-TRAF is a TRAF-binding protein that negatively regulates NF-κB activation. The underlying mechanism of this activity remains unclear. Here we show that TANK directly interacts with PLK1, a conserved cell cycle–regulated kinase. PLK1 inhibits NF-κB transcriptional activation induced by TNF-α, IL-1β, or several activators, but not by nuclear transcription factor p65. PLK1 expression reduces the DNA-binding activity of NF-κB induced by TNF-α. Moreover, endogenous activation of PLK1 reduces the TNF-induced phosphorylation of endogenous IκBα. PLK1 is bound to NEMO (IKKγ) through TANK to form a ternary complex in vivo. We describe a new regulatory mechanism for PLK1: PLK1 negatively regulates TNF-induced IKK activation by inhibiting the ubiquitination of NEMO. These findings reveal that the scaffold protein TANK recruits PLK1 to negatively regulate NF-κB activation and provide direct evidence that PLK1 is required for the repression function of TANK. PMID:20484576

A more detailed picture of the interactions between virtual screening-derived hits and the DNA G-quadruplex: NMR, molecular modelling and ITC studies.

PubMed

Trotta, Roberta; De Tito, Stefano; Lauri, Ilaria; La Pietra, Valeria; Marinelli, Luciana; Cosconati, Sandro; Martino, Luigi; Conte, Maria R; Mayol, Luciano; Novellino, Ettore; Randazzo, Antonio

2011-08-01

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)](4), we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG(Br)GGT)](4) and [d(TGGGG(Br)T)](4). The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)](4) with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

DNA Nanotubes for NMR Structure Determination of Membrane Proteins

PubMed Central

Bellot, Gaëtan; McClintock, Mark A.; Chou, James J; Shih, William M.

2013-01-01

Structure determination of integral membrane proteins by solution NMR represents one of the most important challenges of structural biology. A Residual-Dipolar-Coupling-based refinement approach can be used to solve the structure of membrane proteins up to 40 kDa in size, however, a weak-alignment medium that is detergent-resistant is required. Previously, availability of media suitable for weak alignment of membrane proteins was severely limited. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400nm-long six-helix bundles each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, towards collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes via counter ions and small DNA binding molecules. This detergent-resistant liquid-crystal media offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility, and structural programmability. Production of sufficient nanotubes for 4–5 NMR experiments can be completed in one week by a single individual. PMID:23518667

Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

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

Ding, Fang; Lu, Changrui; Zhao, Wei

2011-07-25

Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 {angstrom} resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNAmore » cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of {psi}29 DNA.« less

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage.

PubMed

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-04-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.

Directing folding pathways for multi-component DNA origami nanostructures with complex topology

NASA Astrophysics Data System (ADS)

Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H.-J.; Castro, C. E.

2016-05-01

Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

DNA nanotubes for NMR structure determination of membrane proteins.

PubMed

Bellot, Gaëtan; McClintock, Mark A; Chou, James J; Shih, William M

2013-04-01

Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.

DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

NASA Astrophysics Data System (ADS)

Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

2011-07-01

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

Hydrogen Peroxide Activated Quinone Methide Precursors with Enhanced DNA Cross-Linking Capability and Cytotoxicity towards Cancer Cells

PubMed Central

Wang, Yibin; Fan, Heli; Balakrishnan, Kumudha; Lin, Zechao; Cao, Sheng; Chen, Wenbing; Fan, Yukai; Guthrie, Quibria A.; Sun, Huabing; Teske, Kelly A.; Gandhi, Varsha; Arnold, Leggy A.; Peng, Xiaohua

2017-01-01

Quinone methide (QM) formation induced by endogenously generated H2O2 is attractive for biological and biomedical applications. To overcome current limitations due to low biological activity of H2O2-activated QM precursors, we are introducing herein several new arylboronates with electron donating substituents at different positions of benzene ring and/or different neutral leaving groups. The reaction rate of the arylboronate esters with H2O2 and subsequent bisquinone methides formation and DNA cross-linking was accelerated with the application of Br as a leaving group instead of acetoxy groups. Additionally, a donating group placed meta to the nascent exo-methylene group of the quinone methide greatly improves H2O2-induced DNA interstrand cross-link formation as well as enhances the cellular activity. Multiple donating groups decrease the stability and DNA cross-linking capability, which lead to low cellular activity. A cell-based screen demonstrated that compounds 2a and 5a with a OMe or OH group dramatically inhibited the growth of various tissue-derived cancer cells while normal cells were less affected. Induction of H2AX phosphorylation by these compounds in CLL lymphocytes provide evidence for a correlation between cell death and DNA damage. The compounds presented herein showed potent anticancer activities and selectivity, which represent a novel scaffold for anticancer drug development. PMID:28388522

Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals

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

Simmons, Chad R.; Zhang, Fei; MacCulloch, Tara

The foundational goal of structural DNA nanotechnology—the field that uses oligonucleotides as a molecular building block for the programmable self-assembly of nanostructured systems—was to use DNA to construct three-dimensional (3D) lattices for solving macromolecular structures. The programmable nature of DNA makes it an ideal system for rationally constructing self-assembled crystals and immobilizing guest molecules in a repeating 3D array through their specific stereospatial interactions with the scaffold. In this work, we have extended a previously described motif (4 × 5) by expanding the structure to a system that links four double-helical layers; we use a central weaving oligonucleotide containing amore » sequence of four six-base repeats (4 × 6), forming a matrix of layers that are organized and dictated by a series of Holliday junctions. In addition, we have assembled mirror image crystals (l-DNA) with the identical sequence that are completely resistant to nucleases. Bromine and selenium derivatives were obtained for the l- and d-DNA forms, respectively, allowing phase determination for both forms and solution of the resulting structures to 3.0 and 3.05 Å resolution. Both right- and left-handed forms crystallized in the trigonal space groups with mirror image 3-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highly organized array of discrete and well-defined cavities that are suitable for hosting guest molecules and allow us to dictate a priori the assembly of guest–DNA conjugates with a specified crystalline hand.« less

PARACEST Properties of a Dinuclear Neodymium(III) Complex Bound to DNA or Carbonate

PubMed Central

Nwe, Kido; Andolina, Christopher M.; Huang, Ching-Hui; Morrow, Janet R.

2009-01-01

A dinuclear Nd(III) macrocyclic complex of 1 (1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene) and mononuclear complexes of 1,4,7-tris-1,4,7,10-tetraazacyclododecane 2, and 1,4,7-tris[(N-N-diethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane, 3, are prepared. Complexes of 1 and 2 give rise to a PARACEST (paramagnetic chemical exchange saturation transfer) peak from exchangeable amide protons that resonate approximately 12 ppm downfield from the bulk water proton resonance. The dinuclear Nd(III) complex is promising as a PARACEST contrast agent for MRI applications because it has an optimal pH of 7.5 and the rate constant for amide proton exchange (2700 s−1) is nearly as large as it can be within slow exchange conditions with bulk water. Dinuclear Ln2(1) complexes (Ln(III) = Nd(III), Eu(III)) bind tightly to anionic ligands including carbonate, diethylphosphate and DNA. The CEST amide peak of Nd2(1) is enhanced by certain DNA sequences that contain hairpin loops, but decreases in the presence of diethyl phosphate or carbonate. Direct excitation luminescence studies of Eu2(1) show that double-stranded and hairpin loop DNA sequences displace one water ligand on each Eu(III) center. DNA displaces carbonate ion despite the low dissociation constant for the Eu2(1) carbonate complex (Kd = 15 µM). Enhancement of the CEST effect of a lanthanide complex by binding to DNA is a promising step toward the preparation of PARACEST agents containing DNA scaffolds. PMID:19555071

PARACEST properties of a dinuclear neodymium(III) complex bound to DNA or carbonate.

PubMed

Nwe, Kido; Andolina, Christopher M; Huang, Ching-Hui; Morrow, Janet R

2009-07-01

A dinuclear Nd(III) macrocyclic complex of 1 (1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene) and mononuclear complexes of 1,4,7-tris-1,4,7,10-tetraazacyclododecane, 2, and 1,4,7-tris[(N-N-diethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane, 3, are prepared. Complexes of 1 and 2 give rise to a PARACEST (paramagnetic chemical exchange saturation transfer) peak from exchangeable amide protons that resonate approximately 12 ppm downfield from the bulk water proton resonance. The dinuclear Nd(III) complex is promising as a PARACEST contrast agent for MRI applications, because it has an optimal pH of 7.5 and the rate constant for amide proton exchange (2700 s(-1)) is nearly as large as it can be within slow exchange conditions with bulk water. Dinuclear Ln(2)(1) complexes (Ln(III) = Nd(III), Eu(III)) bind tightly to anionic ligands including carbonate, diethyl phosphate, and DNA. The CEST amide peak of Nd(2)(1) is enhanced by certain DNA sequences that contain hairpin loops, but decreases in the presence of diethyl phosphate or carbonate. Direct excitation luminescence studies of Eu(2)(1) show that double-stranded and hairpin-loop DNA sequences displace one water ligand on each Eu(III) center. DNA displaces carbonate ion despite the low dissociation constant for the Eu(2)(1) carbonate complex (K(d) = 15 microM). Enhancement of the CEST effect of a lanthanide complex by binding to DNA is a promising step toward the preparation of PARACEST agents containing DNA scaffolds.

DNA materials: bridging nanotechnology and biotechnology.

PubMed

Yang, Dayong; Hartman, Mark R; Derrien, Thomas L; Hamada, Shogo; An, Duo; Yancey, Kenneth G; Cheng, Ru; Ma, Minglin; Luo, Dan

2014-06-17

CONSPECTUS: In recent decades, DNA has taken on an assortment of diverse roles, not only as the central genetic molecule in biological systems but also as a generic material for nanoscale engineering. DNA possesses many exceptional properties, including its biological function, biocompatibility, molecular recognition ability, and nanoscale controllability. Taking advantage of these unique attributes, a variety of DNA materials have been created with properties derived both from the biological functions and from the structural characteristics of DNA molecules. These novel DNA materials provide a natural bridge between nanotechnology and biotechnology, leading to far-ranging real-world applications. In this Account, we describe our work on the design and construction of DNA materials. Based on the role of DNA in the construction, we categorize DNA materials into two classes: substrate and linker. As a substrate, DNA interfaces with enzymes in biochemical reactions, making use of molecular biology's "enzymatic toolkit". For example, employing DNA as a substrate, we utilized enzymatic ligation to prepare the first bulk hydrogel made entirely of DNA. Using this DNA hydrogel as a structural scaffold, we created a protein-producing DNA hydrogel via linking plasmid DNA onto the hydrogel matrix through enzymatic ligation. Furthermore, to fully make use of the advantages of both DNA materials and polymerase chain reaction (PCR), we prepared thermostable branched DNA that could remain intact even under denaturing conditions, allowing for their use as modular primers for PCR. Moreover, via enzymatic polymerization, we have recently constructed a physical DNA hydrogel with unique internal structure and mechanical properties. As a linker, we have used DNA to interface with other functional moieties, including gold nanoparticles, clay minerals, proteins, and lipids, allowing for hybrid materials with unique properties for desired applications. For example, we recently designed a DNA-protein conjugate as a universal adapter for protein detection. We further demonstrate a diverse assortment of applications for these DNA materials including diagnostics, protein production, controlled drug release systems, the exploration of life evolution, and plasmonics. Although DNA has shown great potential as both substrate and linker in the construction of DNA materials, it is still in the initial stages of becoming a well-established and widely used material. Important challenges include the ease of design and fabrication, scaling-up, and minimizing cost. We envision that DNA materials will continue to bridge the gap between nanotechnology and biotechnology and will ultimately be employed for many real-world applications.

Effect of interstitial palladium on plasmon-driven charge transfer in nanoparticle dimers.

PubMed

Lerch, Sarah; Reinhard, Björn M

2018-04-23

Capacitive plasmon coupling between noble metal nanoparticles (NPs) is characterized by an increasing red-shift of the bonding dipolar plasmon mode (BDP) in the classical electromagnetic coupling regime. This model breaks down at short separations where plasmon-driven charge transfer induces a gap current between the NPs with a magnitude and separation dependence that can be modulated if molecules are present in the gap. Here, we use gap contained DNA as a scaffold for the growth of palladium (Pd) NPs in the gap between two gold NPs and investigate the effect of increasing Pd NP concentration on the BDP mode. Consistent with enhanced plasmon-driven charge transfer, the integration of discrete Pd NPs depolarizes the capacitive BDP mode over longer interparticle separations than is possible in only DNA-linked Au NPs. High Pd NP densities in the gap increases the gap conductance and induces the transition from capacitive to conductive coupling.

Shotgun Optical Maps of the Whole Escherichia coli O157:H7 Genome

PubMed Central

Lim, Alex; Dimalanta, Eileen T.; Potamousis, Konstantinos D.; Yen, Galex; Apodoca, Jennifer; Tao, Chunhong; Lin, Jieyi; Qi, Rong; Skiadas, John; Ramanathan, Arvind; Perna, Nicole T.; Plunkett, Guy; Burland, Valerie; Mau, Bob; Hackett, Jeremiah; Blattner, Frederick R.; Anantharaman, Thomas S.; Mishra, Bhubaneswar; Schwartz, David C.

2001-01-01

We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies. PMID:11544203

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis.

PubMed

Shao, Changwei; Niu, Yongchao; Rastas, Pasi; Liu, Yang; Xie, Zhiyuan; Li, Hengde; Wang, Lei; Jiang, Yong; Tai, Shuaishuai; Tian, Yongsheng; Sakamoto, Takashi; Chen, Songlin

2015-04-01

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1-8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Tying a molecular knot with optical tweezers

NASA Astrophysics Data System (ADS)

Arai, Yasuharu; Yasuda, Ryohei; Akashi, Ken-Ichirou; Harada, Yoshie; Miyata, Hidetake; Kinosita, Kazuhiko; Itoh, Hiroyasu

1999-06-01

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility-for example, DNA-binding proteins can induce sharp bends in the helix. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions or of the response to external forces or flows. Controlled buckling of a microtubule has been reported, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strand's ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4µm. The pulling force at breakage is around 1pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.

Enhanced gene delivery to the lung using biodegradable polyunsaturated cationic phosphatidylcholine-detergent conjugates.

PubMed

Pierrat, Philippe; Kereselidze, Dimitri; Lux, Marie; Lebeau, Luc; Pons, Françoise

2016-09-10

Lung diseases are among the more representative causes of mortality and morbidity worldwide and gene therapy is considered as a promising therapeutic approach for their treatment. However the design of efficient nucleic acid carriers for airway administration still is a challenge and there is a pressing need for new developments in this field. Herein, new synthetic DNA carriers based on the conjugation of a phospholipid and C12E4, a nonionic detergent, are developed. DNA complexes with phosphatidylcholine-detergent conjugates are administered in mouse airways, and transgene expression and inflammatory activity as an index of toxicity are investigated as a function of time, DNA dose, and presence of helper and stealth lipids. Introduction of a biodegradable linker between the phosphatidylcholine and detergent moieties significantly attenuates the severity of inflammatory response that characterizes cationic lipid-mediated gene transfer. Concurrent introduction of polyunsaturated fatty acid chains in the carrier scaffold improves transgene expression and further reduces airway inflammation. Finally, the biodegradable phosphatidylcholine-detergent conjugates favorably compare to GL67A, the gold standard for DNA delivery to the airway that is currently under clinical evaluation. Our findings indicate that the lipid formulations described herein may have great potential as nucleic acid carriers for gene therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Terminating DNA Tile Assembly with Nanostructured Caps.

PubMed

Agrawal, Deepak K; Jiang, Ruoyu; Reinhart, Seth; Mohammed, Abdul M; Jorgenson, Tyler D; Schulman, Rebecca

2017-10-24

Precise control over the nucleation, growth, and termination of self-assembly processes is a fundamental tool for controlling product yield and assembly dynamics. Mechanisms for altering these processes programmatically could allow the use of simple components to self-assemble complex final products or to design processes allowing for dynamic assembly or reconfiguration. Here we use DNA tile self-assembly to develop general design principles for building complexes that can bind to a growing biomolecular assembly and terminate its growth by systematically characterizing how different DNA origami nanostructures interact with the growing ends of DNA tile nanotubes. We find that nanostructures that present binding interfaces for all of the binding sites on a growing facet can bind selectively to growing ends and stop growth when these interfaces are presented on either a rigid or floppy scaffold. In contrast, nucleation of nanotubes requires the presentation of binding sites in an arrangement that matches the shape of the structure's facet. As a result, it is possible to build nanostructures that can terminate the growth of existing nanotubes but cannot nucleate a new structure. The resulting design principles for constructing structures that direct nucleation and termination of the growth of one-dimensional nanostructures can also serve as a starting point for programmatically directing two- and three-dimensional crystallization processes using nanostructure design.

Competitive annealing of multiple DNA origami: formation of chimeric origami

NASA Astrophysics Data System (ADS)

Majikes, Jacob M.; Nash, Jessica A.; LaBean, Thomas H.

2016-11-01

Scaffolded DNA origami are a robust tool for building discrete nanoscale objects at high yield. This strategy ensures, in the design process, that the desired nanostructure is the minimum free energy state for the designed set of DNA sequences. Despite aiming for the minimum free energy structure, the folding process which leads to that conformation is difficult to characterize, although it has been the subject of much research. In order to shed light on the molecular folding pathways, this study intentionally frustrates the folding process of these systems by simultaneously annealing the staple pools for multiple target or parent origami structures, forcing competition. A surprising result of these competitive, simultaneous anneals is the formation of chimeric DNA origami which inherit structural regions from both parent origami. By comparing the regions inherited from the parent origami, relative stability of substructures were compared. This allowed examination of the folding process with typical characterization techniques and materials. Anneal curves were then used as a means to rapidly generate a phase diagram of anticipated behavior as a function of staple excess and parent staple ratio. This initial study shows that competitive anneals provide an exciting way to create diverse new nanostructures and may be used to examine the relative stability of various structural motifs.

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

PubMed Central

Liu, Jun; Zimmer, Kurt; Rusch, Douglas B.; Paranjape, Neha; Podicheti, Ram; Tang, Haixu; Calvi, Brian R.

2015-01-01

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC. PMID:26227968

Integrated Nanosystems Templated by Self-assembled Virus Capsids

NASA Astrophysics Data System (ADS)

Stephanopoulos, Nicholas

This dissertation presents the synthesis and modeling of multicomponent nanosystems templated by self-assembled virus capsids. The design principles, synthesis, analysis, and future directions for these capsid-based materials are presented. Chapter 1 gives an overview of the literature on the application of virus capsids in constructing nanomaterials. The uses of capsids in three main areas are considered: (1) as templates for inorganic materials or nanoparticles; (2) as vehicles for biological applications like medical imaging and treatment; and (3) as scaffolds for catalytic materials. In light of this introduction, an overview of the material in this dissertation is described. Chapters 2-4 all describe integrated nanosystems templated by bacteriophage MS2, a spherical icosahedral virus capsid. MS2 possesses an interior and exterior surface that can be modified orthogonally using bioconjugation chemistry to create multivalent, multicomponent constructs with precise localization of components attached to the capsid proteins. Chapter 2 describes the use of MS2 to synthesize a photocatalytic construct by modifying the internal surface with sensitizing chromophores and the external surface with a photocatalytic porphyrin. The chromophores absorbed energy that the porphyrin could not, and transferred it to the porphyrin via FRET through the protein shell. The porphyrin was then able to utilize the energy to carry out photocatalysis at new wavelengths. In Chapter 3, porphyrins were installed on the interior surface of MS2 and DNA aptamers specific for Jurkat leukemia T cells on the exterior surface. The dual-modified capsids were able to bind to Jurkat cells, and upon illumination the porphyrins generated singlet oxygen to kill them selectively over non-targeted cells. Chapter 4 explores integrating MS2 with DNA origami in order to arrange the capsids at larger length scales. Capsids modified with fluorescent dyes inside and single-stranded DNA outside were able to bind to origami tiles bearing complementary DNA probes. The tiles could then be used to arrange the capsids in a one-dimensional array with dimensions far exceeding those of individual MS2 particles. In Chapter 5, the use of a different capsid, that of the tobacco mosaic virus (TMV) is described. The defect tolerance of light harvesting systems built using TMV as a scaffold was investigated using a kinetic Monte Carlo model to simulate the energy transfer processes. The results of the simulation were used to understand and explain experimental results obtained from the system.

Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering.

PubMed

Haaparanta, Anne-Marie; Järvinen, Elina; Cengiz, Ibrahim Fatih; Ellä, Ville; Kokkonen, Harri T; Kiviranta, Ilkka; Kellomäki, Minna

2014-04-01

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

Comparison of the properties of collagen-chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking.

PubMed

Chen, Zihao; Du, Tianming; Tang, Xiangyu; Liu, Changjun; Li, Ruixin; Xu, Cheng; Tian, Feng; Du, Zhenjie; Wu, Jimin

2016-07-01

The property of collagen-chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen-chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen-chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen-chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.

SHOP: scaffold HOPping by GRID-based similarity searches.

PubMed

Bergmann, Rikke; Linusson, Anna; Zamora, Ismael

2007-05-31

A new GRID-based method for scaffold hopping (SHOP) is presented. In a fully automatic manner, scaffolds were identified in a database based on three types of 3D-descriptors. SHOP's ability to recover scaffolds was assessed and validated by searching a database spiked with fragments of known ligands of three different protein targets relevant for drug discovery using a rational approach based on statistical experimental design. Five out of eight and seven out of eight thrombin scaffolds and all seven HIV protease scaffolds were recovered within the top 10 and 31 out of 31 neuraminidase scaffolds were in the 31 top-ranked scaffolds. SHOP also identified new scaffolds with substantially different chemotypes from the queries. Docking analysis indicated that the new scaffolds would have similar binding modes to those of the respective query scaffolds observed in X-ray structures. The databases contained scaffolds from published combinatorial libraries to ensure that identified scaffolds could be feasibly synthesized.

[Three-dimensional parallel collagen scaffold promotes tendon extracellular matrix formation].

PubMed

Zheng, Zefeng; Shen, Weiliang; Le, Huihui; Dai, Xuesong; Ouyang, Hongwei; Chen, Weishan

2016-03-01

To investigate the effects of three-dimensional parallel collagen scaffold on the cell shape, arrangement and extracellular matrix formation of tendon stem cells. Parallel collagen scaffold was fabricated by unidirectional freezing technique, while random collagen scaffold was fabricated by freeze-drying technique. The effects of two scaffolds on cell shape and extracellular matrix formation were investigated in vitro by seeding tendon stem/progenitor cells and in vivo by ectopic implantation. Parallel and random collagen scaffolds were produced successfully. Parallel collagen scaffold was more akin to tendon than random collagen scaffold. Tendon stem/progenitor cells were spindle-shaped and unified orientated in parallel collagen scaffold, while cells on random collagen scaffold had disorder orientation. Two weeks after ectopic implantation, cells had nearly the same orientation with the collagen substance. In parallel collagen scaffold, cells had parallel arrangement, and more spindly cells were observed. By contrast, cells in random collagen scaffold were disorder. Parallel collagen scaffold can induce cells to be in spindly and parallel arrangement, and promote parallel extracellular matrix formation; while random collagen scaffold can induce cells in random arrangement. The results indicate that parallel collagen scaffold is an ideal structure to promote tendon repairing.

A switchable positive and negative air pressure device for efficient and gentle handling of nanofiber scaffolds

NASA Astrophysics Data System (ADS)

Hotaling, Nathan A.; Khristov, Vladimir; Maminishkis, Arvydas; Bharti, Kapil; Simon, Carl G.

2017-10-01

A scaffold handling device (SHD) has been designed that can switch from gentle suction to positive pressure to lift and place nanofiber scaffolds. In tissue engineering laboratories, delicate fibrous scaffolds, such as electrospun nanofiber scaffolds, are often used as substrates for cell culture. Typical scaffold handling procedures include lifting the scaffolds, moving them from one container to another, sterilization, and loading scaffolds into cell culture plates. Using tweezers to handle the scaffolds can be slow, can damage the scaffolds, and can cause them to wrinkle or fold. Scaffolds may also acquire a static charge which makes them difficult to put down as they cling to tweezers. An SHD has been designed that enables more efficient, gentle lifting, and placement of delicate scaffolds. Most of the parts to make the SHD can be purchased, except for the tip which can be 3D-printed. The SHD enables more reliable handling of nanofiber scaffolds that may improve the consistency of biomanufacturing processes.

Mouse embryonic stem cells have increased capacity for replication fork restart driven by the specific Filia-Floped protein complex.

PubMed

Zhao, Bo; Zhang, Weidao; Cun, Yixian; Li, Jingzheng; Liu, Yan; Gao, Jing; Zhu, Hongwen; Zhou, Hu; Zhang, Rugang; Zheng, Ping

2018-01-01

Pluripotent stem cells (PSCs) harbor constitutive DNA replication stress during their rapid proliferation and the consequent genome instability hampers their applications in regenerative medicine. It is therefore important to understand the regulatory mechanisms of replication stress response in PSCs. Here, we report that mouse embryonic stem cells (ESCs) are superior to differentiated cells in resolving replication stress. Specifically, ESCs utilize a unique Filia-Floped protein complex-dependent mechanism to efficiently promote the restart of stalled replication forks, therefore maintaining genomic stability. The ESC-specific Filia-Floped complex resides on replication forks under normal conditions. Replication stress stimulates their recruitment to stalling forks and the serine 151 residue of Filia is phosphorylated in an ATR-dependent manner. This modification enables the Filia-Floped complex to act as a functional scaffold, which then promotes the stalling fork restart through a dual mechanism: both enhancing recruitment of the replication fork restart protein, Blm, and stimulating ATR kinase activation. In the Blm pathway, the scaffolds recruit the E3 ubiquitin ligase, Trim25, to the stalled replication forks, and in turn Trim25 tethers and concentrates Blm at stalled replication forks through ubiquitination. In differentiated cells, the recruitment of the Trim25-Blm complex to replication forks and the activation of ATR signaling are much less robust due to lack of the ESC-specific Filia-Floped scaffold. Thus, our study reveals that ESCs utilize an additional and unique regulatory layer to efficiently promote the stalled fork restart and maintain genomic stability.

A RAD-Based Genetic Map for Anchoring Scaffold Sequences and Identifying QTLs in Bitter Gourd (Momordica charantia)

PubMed Central

Cui, Junjie; Luo, Shaobo; Niu, Yu; Huang, Rukui; Wen, Qingfang; Su, Jianwen; Miao, Nansheng; He, Weiming; Dong, Zhensheng; Cheng, Jiaowen; Hu, Kailin

2018-01-01

Genetic mapping is a basic tool necessary for anchoring assembled scaffold sequences and for identifying QTLs controlling important traits. Though bitter gourd (Momordica charantia) is both consumed and used as a medicinal, research on its genomics and genetic mapping is severely limited. Here, we report the construction of a restriction site associated DNA (RAD)-based genetic map for bitter gourd using an F2 mapping population comprising 423 individuals derived from two cultivated inbred lines, the gynoecious line ‘K44’ and the monoecious line ‘Dali-11.’ This map comprised 1,009 SNP markers and spanned a total genetic distance of 2,203.95 cM across the 11 linkage groups. It anchored a total of 113 assembled scaffolds that covered about 251.32 Mb (85.48%) of the 294.01 Mb assembled genome. In addition, three horticulturally important traits including sex expression, fruit epidermal structure, and immature fruit color were evaluated using a combination of qualitative and quantitative data. As a result, we identified three QTL/gene loci responsible for these traits in three environments. The QTL/gene gy/fffn/ffn, controlling sex expression involved in gynoecy, first female flower node, and female flower number was detected in the reported region. Particularly, two QTLs/genes, Fwa/Wr and w, were found to be responsible for fruit epidermal structure and white immature fruit color, respectively. This RAD-based genetic map promotes the assembly of the bitter gourd genome and the identified genetic loci will accelerate the cloning of relevant genes in the future. PMID:29706980

Efficient identification of Y chromosome sequences in the human and Drosophila genomes.

PubMed

Carvalho, Antonio Bernardo; Clark, Andrew G

2013-11-01

Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes.

Force-Induced Unravelling of DNA Origami.

PubMed

Engel, Megan C; Smith, David M; Jobst, Markus A; Sajfutdinow, Martin; Liedl, Tim; Romano, Flavio; Rovigatti, Lorenzo; Louis, Ard A; Doye, Jonathan P K

2018-05-31

The mechanical properties of DNA nanostructures are of widespread interest as applications that exploit their stability under constant or intermittent external forces become increasingly common. We explore the force response of DNA origami in comprehensive detail by combining AFM single molecule force spectroscopy experiments with simulations using oxDNA, a coarse-grained model of DNA at the nucleotide level, to study the unravelling of an iconic origami system: the Rothemund tile. We contrast the force-induced melting of the tile with simulations of an origami 10-helix bundle. Finally, we simulate a recently-proposed origami biosensor, whose function takes advantage of origami behaviour under tension. We observe characteristic stick-slip unfolding dynamics in our force-extension curves for both the Rothemund tile and the helix bundle and reasonable agreement with experimentally observed rupture forces for these systems. Our results highlight the effect of design on force response: we observe regular, modular unfolding for the Rothemund tile that contrasts with strain-softening of the 10-helix bundle which leads to catastropic failure under monotonically increasing force. Further, unravelling occurs straightforwardly from the scaffold ends inwards for the Rothemund tile, while the helix bundle unfolds more nonlinearly. The detailed visualization of the yielding events provided by simulation allows preferred pathways through the complex unfolding free-energy landscape to be mapped, as a key factor in determining relative barrier heights is the extensional release per base pair broken. We shed light on two important questions: how stable DNA nanostructures are under external forces; and what design principles can be applied to enhance stability.

Controlled superficial assembly of DNA-amorphous calcium phosphate nanocomposite spheres for surface-mediated gene delivery.

PubMed

Oyane, Ayako; Araki, Hiroko; Nakamura, Maki; Shimizu, Yoshiki; Shubhra, Quazi T H; Ito, Atsuo; Tsurushima, Hideo

2016-05-01

Surface-mediated gene delivery systems have many potential applications in tissue engineering. We recently fabricated an assembly consisting of DNA-amorphous calcium phosphate (DNA-ACP) nanocomposite spheres on a polymer substrate via coprecipitation in a labile supersaturated calcium phosphate (CaP) solution and demonstrated the assembly's high gene delivery efficacy. In this study, we conducted a detailed investigation of the coprecipitation process in solution and revealed that the negatively charged DNA molecules were immobilized in the ACP spheres during the initial stage of coprecipitation and functioned as both sphere-dispersing and size-regulating agents. As a result, the DNA-ACP nanocomposites grew into size-regulated submicrospheres in solution and assembled onto the substrate via gravity sedimentation. The assembled nanocomposite spheres were chemically anchored to the substrate surface through an intermediate layer of CaP-based nanoparticles that was formed heterogeneously at the substrate surface. The coprecipitation conditions, i.e., coprecipitation time and Ca and P concentrations in solution, greatly affected the state of assembly of the nanocomposite spheres, thereby influencing the gene expression level of the cells cultured on the substrate. Increasing the number density and decreasing the size of the nanocomposite spheres did not always increase the assembly's gene delivery efficacy (per surface area of the substrate) due to adverse effects on cellular viability. As demonstrated herein, controlling the coprecipitation conditions is important for designing a cell-stimulating and biocompatible scaffold surface consisting of an assembly of DNA-ACP nanocomposite spheres. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy.

PubMed

Wang, Xiaoying; Shu, Guofang; Gao, Chanchan; Yang, Yu; Xu, Qian; Tang, Meng

2014-12-01

An electrochemical biosensor based on functional composite nanofibers for hybridization detection of specific K-ras gene that is highly associated with colorectal cancer via multiple signal amplification strategy has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped nylon 6 (PA6) composite nanofibers (MWCNTs-PA6) was prepared using electrospinning, which served as the nanosized backbone for thionine (TH) electropolymerization. The functional composite nanofibers [MWCNTs-PA6-PTH, where PTH is poly(thionine)] used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the hybridization sensitivity. Through the hybridization reaction, a sandwich format of ssDNA1/K-ras gene/gold nanoparticle-labeled ssDNA2 (AuNPs-ssDNA2) was fabricated, and the AuNPs offered excellent electrochemical signal transduction. The signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles (TA/AuNPs). A significant sensitivity enhancement was obtained; the detection limit was down to 30fM, and the discriminations were up to 54.3 and 51.9% between the K-ras gene and the one-base mismatched sequences including G/C and A/T mismatched bases, respectively. The amenability of this method to the analyses of K-ras gene from the SW480 colorectal cancer cell lysates was demonstrated. The results are basically consistent with those of the K-ras Kit (HRM: high-resolution melt). The method holds promise for the diagnosis and management of cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

A nucleotide-analogue-induced gain of function corrects the error-prone nature of human DNA polymerase iota.

PubMed

Ketkar, Amit; Zafar, Maroof K; Banerjee, Surajit; Marquez, Victor E; Egli, Martin; Eoff, Robert L

2012-06-27

Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2'-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2'-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.

A nucleotide analogue induced gain of function corrects the error-prone nature of human DNA polymerase iota

PubMed Central

Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L

2012-01-01

Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase. PMID:22632140

Spiral-structured, nanofibrous, 3D scaffolds for bone tissue engineering.

PubMed

Wang, Junping; Valmikinathan, Chandra M; Liu, Wei; Laurencin, Cato T; Yu, Xiaojun

2010-05-01

Polymeric nanofiber matrices have already been widely used in tissue engineering. However, the fabrication of nanofibers into complex three-dimensional (3D) structures is restricted due to current manufacturing techniques. To overcome this limitation, we have incorporated nanofibers onto spiral-structured 3D scaffolds made of poly (epsilon-caprolactone) (PCL). The spiral structure with open geometries, large surface areas, and porosity will be helpful for improving nutrient transport and cell penetration into the scaffolds, which are otherwise limited in conventional tissue-engineered scaffolds for large bone defects repair. To investigate the effect of structure and fiber coating on the performance of the scaffolds, three groups of scaffolds including cylindrical PCL scaffolds, spiral PCL scaffolds (without fiber coating), and spiral-structured fibrous PCL scaffolds (with fiber coating) have been prepared. The morphology, porosity, and mechanical properties of the scaffolds have been characterized. Furthermore, human osteoblast cells are seeded on these scaffolds, and the cell attachment, proliferation, differentiation, and mineralized matrix deposition on the scaffolds are evaluated. The results indicated that the spiral scaffolds possess porosities within the range of human trabecular bone and an appropriate pore structure for cell growth, and significantly lower compressive modulus and strength than cylindrical scaffolds. When compared with the cylindrical scaffolds, the spiral-structured scaffolds demonstrated enhanced cell proliferation, differentiation, and mineralization and allowed better cellular growth and penetration. The incorporation of nanofibers onto spiral scaffolds further enhanced cell attachment, proliferation, and differentiation. These studies suggest that spiral-structured nanofibrous scaffolds may serve as promising alternatives for bone tissue engineering applications. Copyright 2009 Wiley Periodicals, Inc.

Systematic assessment of scaffold hopping versus activity cliff formation across bioactive compound classes following a molecular hierarchy.

PubMed

Stumpfe, Dagmar; Dimova, Dilyana; Bajorath, Jürgen

2015-07-01

Scaffold hopping and activity cliff formation define opposite ends of the activity landscape feature spectrum. To rationalize these events at the level of scaffolds, active compounds involved in scaffold hopping were required to contain topologically distinct scaffolds but have only limited differences in potency, whereas compounds involved in activity cliffs were required to share the same scaffold but have large differences in potency. A systematic search was carried out for compounds involved in scaffold hopping and/or activity cliff formation. Results obtained for compound data sets covering more than 300 human targets revealed clear trends. If scaffolds represented multiple but fewer than 10 active compounds, nearly 90% of all scaffolds were exclusively involved in hopping events. With increasing compound coverage, the fraction of scaffolds involved in both scaffold hopping and activity cliff formation significantly increased to more than 50%. However, ∼40% of the scaffolds representing large numbers of active compounds continued to be exclusively involved in scaffold hopping. More than 200 scaffolds with broad target coverage were identified that consistently represented potent compounds and yielded an abundance of scaffold hops in the low-nanomolar range. These and other subsets of scaffolds we characterized are of prime interest for structure-activity relationship (SAR) exploration and compound design. Therefore, the complete scaffold classification generated in the course of our analysis is made freely available. Copyright © 2015 Elsevier Ltd. All rights reserved.

Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

PubMed

Zhang, Qiang; Xing, Hui-Li; Wang, Zhi-Ping; Zhang, Hai-Yan; Yang, Fang; Wang, Xue-Chen; Chen, Qi-Jun

2018-03-01

We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

N-Heterocyclic choline analogues based on 1,2,3,4-tetrahydro(iso)quinoline scaffold with anticancer and anti-infective dual action.

PubMed

Zablotskaya, Alla; Segal, Izolda; Geronikaki, Athina; Shestakova, Irina; Nikolajeva, Vizma; Makarenkova, Galina

2017-06-01

Pharmacological effects of biologically active "small molecules" can be improved by their targeted modification, which affects drug delivery and interaction with tumor cells and microorganisms. We aimed to evaluate anticancer and antimicrobial activity of lipid-like choline derivatives modified via simultaneous introduction of tetrahydro(iso)quinoline based pharmacophore system at nitrogen atom and long chain alkyl substituent at oxygen atom. Target compounds were synthesized under phase-transfer catalysis conditions followed by quaternization, and evaluated for cytotoxicity and NO-generation ability on HT-1080 and MG-22A tumor cell lines and NIH 3T3 normal mouse fibroblasts, and screened for antimicrobial activity against gram-positive (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis) and fungi (Candida albicans and Aspergillus niger). Inhibitory action of active compounds towards E. coli DNA gyrase was investigated. Target compounds exhibit high selective cytotoxicity (LC 50 <1μg/mL) and NO-induction ability, and reveal strong antimicrobial activity with MIC and MBC/MFC values of 0.5-32μg/mL, predominantly vs. gram-positive bacteria and fungi. Tested substances displayed inhibitory effect towards E. coli DNA gyrase, though less than ciprofloxacin. Tetrahydroisoquinoline derivatives and compounds possessing substituents with chain length of 10 and 11 carbon atoms have highest indices of activities. Lipid-like N-heterocyclic choline analogues based on 1,2,3,4-tetrahydro(iso)quinoline scaffold, possessing very high cytotoxicity with attendant strong antimicrobial activity are the leads for developing effective dual action therapeutics. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Development of hybrid scaffolds using ceramic and hydrogel for articular cartilage tissue regeneration.

PubMed

Seol, Young-Joon; Park, Ju Young; Jeong, Wonju; Kim, Tae-Ho; Kim, Shin-Yoon; Cho, Dong-Woo

2015-04-01

The regeneration of articular cartilage consisting of hyaline cartilage and hydrogel scaffolds has been generally used in tissue engineering. However, success in in vivo studies has been rarely reported. The hydrogel scaffolds implanted into articular cartilage defects are mechanically unstable and it is difficult for them to integrate with the surrounding native cartilage tissue. Therefore, it is needed to regenerate cartilage and bone tissue simultaneously. We developed hybrid scaffolds with hydrogel scaffolds for cartilage tissue and with ceramic scaffolds for bone tissue. For in vivo study, hybrid scaffolds were press-fitted into osteochondral tissue defects in a rabbit knee joints and the cartilage tissue regeneration in blank, hydrogel scaffolds, and hybrid scaffolds was compared. In 12th week after implantation, the histological and immunohistochemical analyses were conducted to evaluate the cartilage tissue regeneration. In the blank and hydrogel scaffold groups, the defects were filled with fibrous tissues and the implanted hydrogel scaffolds could not maintain their initial position; in the hybrid scaffold group, newly generated cartilage tissues were morphologically similar to native cartilage tissues and were smoothly connected to the surrounding native tissues. This study demonstrates hybrid scaffolds containing hydrogel and ceramic scaffolds can provide mechanical stability to hydrogel scaffolds and enhance cartilage tissue regeneration at the defect site. © 2014 Wiley Periodicals, Inc.

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage

PubMed Central

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-01-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence that CRL4Cdt2-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4Cdt2 as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage. PMID:24423875

Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

DOE PAGES

Aceytuno, R.  Daniel; Piett, Cortt G.; Havali-Shahriari, Zahra; ...

2017-04-27

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5'-phosphate/3'-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation andmore » that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexiblemultistate complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. Amutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.« less

High-Throughput Identification of Combinatorial Ligands for DNA Delivery in Cell Culture

NASA Astrophysics Data System (ADS)

Svahn, Mathias G.; Rabe, Kersten S.; Barger, Geoffrey; EL-Andaloussi, Samir; Simonson, Oscar E.; Didier, Boturyn; Olivier, Renaudet; Dumy, Pascal; Brandén, Lars J.; Niemeyer, Christof M.; Smith, C. I. Edvard

2008-10-01

Finding the optimal combinations of ligands for tissue-specific delivery is tedious even if only a few well-established compounds are tested. The cargo affects the receptor-ligand interaction, especially when it is charged like DNA. The ligand should therefore be evaluated together with its cargo. Several viruses have been shown to interact with more than one receptor, for efficient internalization. We here present a DNA oligonucleotide-based method for inexpensive and rapid screening of biotin labeled ligands for combinatorial effects on cellular binding and uptake. The oligonucleotide complex was designed as a 44 bp double-stranded DNA oligonucleotide with one central streptavidin molecule and a second streptavidin at the terminus. The use of a highly advanced robotic platform ensured stringent processing and execution of the experiments. The oligonucleotides were fluorescently labeled and used for detection and analysis of cell-bound, internalized and intra-cellular compartmentalized constructs by an automated line-scanning confocal microscope, IN Cell Analyzer 3000. All possible combinations of 22 ligands were explored in sets of 2 and tested on 6 different human cell lines in triplicates. In total, 10 000 transfections were performed on the automation platform. Cell-specific combinations of ligands were identified and their relative position on the scaffold oligonucleotide was found to be of importance. The ligands were found to be cargo dependent, carbohydrates were more potent for DNA delivery whereas cell penetrating peptides were more potent for delivery of less charged particles.

Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

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

Aceytuno, R.  Daniel; Piett, Cortt G.; Havali-Shahriari, Zahra

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5'-phosphate/3'-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation andmore » that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexiblemultistate complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. Amutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.« less

Characterization and evaluation of graphene oxide scaffold for periodontal wound healing of class II furcation defects in dog.

PubMed

Kawamoto, Kohei; Miyaji, Hirofumi; Nishida, Erika; Miyata, Saori; Kato, Akihito; Tateyama, Akito; Furihata, Tomokazu; Shitomi, Kanako; Iwanaga, Toshihiko; Sugaya, Tsutomu

2018-01-01

The 3-dimensional scaffold plays a key role in volume and quality of repair tissue in periodontal tissue engineering therapy. We fabricated a novel 3D collagen scaffold containing carbon-based 2-dimensional layered material, named graphene oxide (GO). The aim of this study was to characterize and assess GO scaffold for periodontal tissue healing of class II furcation defects in dog. GO scaffolds were prepared by coating the surface of a 3D collagen sponge scaffold with GO dispersion. Scaffolds were characterized using cytotoxicity and tissue reactivity tests. In addition, GO scaffold was implanted into dog class II furcation defects and periodontal healing was investigated at 4 weeks postsurgery. GO scaffold exhibited low cytotoxicity and enhanced cellular ingrowth behavior and rat bone forming ability. In addition, GO scaffold stimulated healing of dog class II furcation defects. Periodontal attachment formation, including alveolar bone, periodontal ligament-like tissue, and cementum-like tissue, was significantly increased by GO scaffold implantation, compared with untreated scaffold. The results suggest that GO scaffold is biocompatible and possesses excellent bone and periodontal tissue formation ability. Therefore, GO scaffold would be beneficial for periodontal tissue engineering therapy.

Defining scaffold geometries for interacting with proteins: geometrical classification of secondary structure linking regions.

PubMed

Tran, Tran T; Kulis, Christina; Long, Steven M; Bryant, Darryn; Adams, Peter; Smythe, Mark L

2010-11-01

Medicinal chemists synthesize arrays of molecules by attaching functional groups to scaffolds. There is evidence suggesting that some scaffolds yield biologically active molecules more than others, these are termed privileged substructures. One role of the scaffold is to present its side-chains for molecular recognition, and biologically relevant scaffolds may present side-chains in biologically relevant geometries or shapes. Since drug discovery is primarily focused on the discovery of compounds that bind to proteinaceous targets, we have been deciphering the scaffold shapes that are used for binding proteins as they reflect biologically relevant shapes. To decipher the scaffold architecture that is important for binding protein surfaces, we have analyzed the scaffold architecture of protein loops, which are defined in this context as continuous four residue segments of a protein chain that are not part of an α-helix or β-strand secondary structure. Loops are an important molecular recognition motif of proteins. We have found that 39 clusters reflect the scaffold architecture of 89% of the 23,331 loops in the dataset, with average intra-cluster and inter-cluster RMSD of 0.47 and 1.91, respectively. These protein loop scaffolds all have distinct shapes. We have used these 39 clusters that reflect the scaffold architecture of protein loops as biological descriptors. This involved generation of a small dataset of scaffold-based peptidomimetics. We found that peptidomimetic scaffolds with reported biological activities matched loop scaffold geometries and those peptidomimetic scaffolds with no reported biologically activities did not. This preliminary evidence suggests that organic scaffolds with tight matches to the preferred loop scaffolds of proteins, implies the likelihood of the scaffold to be biologically relevant.

Defining scaffold geometries for interacting with proteins: geometrical classification of secondary structure linking regions

NASA Astrophysics Data System (ADS)

Tran, Tran T.; Kulis, Christina; Long, Steven M.; Bryant, Darryn; Adams, Peter; Smythe, Mark L.

2010-11-01

Medicinal chemists synthesize arrays of molecules by attaching functional groups to scaffolds. There is evidence suggesting that some scaffolds yield biologically active molecules more than others, these are termed privileged substructures. One role of the scaffold is to present its side-chains for molecular recognition, and biologically relevant scaffolds may present side-chains in biologically relevant geometries or shapes. Since drug discovery is primarily focused on the discovery of compounds that bind to proteinaceous targets, we have been deciphering the scaffold shapes that are used for binding proteins as they reflect biologically relevant shapes. To decipher the scaffold architecture that is important for binding protein surfaces, we have analyzed the scaffold architecture of protein loops, which are defined in this context as continuous four residue segments of a protein chain that are not part of an α-helix or β-strand secondary structure. Loops are an important molecular recognition motif of proteins. We have found that 39 clusters reflect the scaffold architecture of 89% of the 23,331 loops in the dataset, with average intra-cluster and inter-cluster RMSD of 0.47 and 1.91, respectively. These protein loop scaffolds all have distinct shapes. We have used these 39 clusters that reflect the scaffold architecture of protein loops as biological descriptors. This involved generation of a small dataset of scaffold-based peptidomimetics. We found that peptidomimetic scaffolds with reported biological activities matched loop scaffold geometries and those peptidomimetic scaffolds with no reported biologically activities did not. This preliminary evidence suggests that organic scaffolds with tight matches to the preferred loop scaffolds of proteins, implies the likelihood of the scaffold to be biologically relevant.

Drug-likeness analysis of traditional Chinese medicines: 2. Characterization of scaffold architectures for drug-like compounds, non-drug-like compounds, and natural compounds from traditional Chinese medicines.

PubMed

Tian, Sheng; Li, Youyong; Wang, Junmei; Xu, Xiaojie; Xu, Lei; Wang, Xiaohong; Chen, Lei; Hou, Tingjun

2013-01-21

In order to better understand the structural features of natural compounds from traditional Chinese medicines, the scaffold architectures of drug-like compounds in MACCS-II Drug Data Report (MDDR), non-drug-like compounds in Available Chemical Directory (ACD), and natural compounds in Traditional Chinese Medicine Compound Database (TCMCD) were explored and compared. First, the different scaffolds were extracted from ACD, MDDR and TCMCD by using three scaffold representations, including Murcko frameworks, Scaffold Tree, and ring systems with different complexity and side chains. Then, by examining the accumulative frequency of the scaffolds in each dataset, we observed that the Level 1 scaffolds of the Scaffold Tree offer advantages over the other scaffold architectures to represent the scaffold diversity of the compound libraries. By comparing the similarity of the scaffold architectures presented in MDDR, ACD and TCMCD, structural overlaps were observed not only between MDDR and TCMCD but also between MDDR and ACD. Finally, Tree Maps were used to cluster the Level 1 scaffolds of the Scaffold Tree and visualize the scaffold space of the three datasets. The analysis of the scaffold architectures of MDDR, ACD and TCMCD shows that, on average, drug-like molecules in MDDR have the highest diversity while natural compounds in TCMCD have the highest complexity. According to the Tree Maps, it can be observed that the Level 1 scaffolds present in MDDR have higher diversity than those presented in TCMCD and ACD. However, some representative scaffolds in MDDR with high frequency show structural similarities to those in TCMCD and ACD, suggesting that some scaffolds in TCMCD and ACD may be potentially drug-like fragments for fragment-based and de novo drug design.

Drug-likeness analysis of traditional Chinese medicines: 2. Characterization of scaffold architectures for drug-like compounds, non-drug-like compounds, and natural compounds from traditional Chinese medicines

PubMed Central

2013-01-01

Background In order to better understand the structural features of natural compounds from traditional Chinese medicines, the scaffold architectures of drug-like compounds in MACCS-II Drug Data Report (MDDR), non-drug-like compounds in Available Chemical Directory (ACD), and natural compounds in Traditional Chinese Medicine Compound Database (TCMCD) were explored and compared. Results First, the different scaffolds were extracted from ACD, MDDR and TCMCD by using three scaffold representations, including Murcko frameworks, Scaffold Tree, and ring systems with different complexity and side chains. Then, by examining the accumulative frequency of the scaffolds in each dataset, we observed that the Level 1 scaffolds of the Scaffold Tree offer advantages over the other scaffold architectures to represent the scaffold diversity of the compound libraries. By comparing the similarity of the scaffold architectures presented in MDDR, ACD and TCMCD, structural overlaps were observed not only between MDDR and TCMCD but also between MDDR and ACD. Finally, Tree Maps were used to cluster the Level 1 scaffolds of the Scaffold Tree and visualize the scaffold space of the three datasets. Conclusion The analysis of the scaffold architectures of MDDR, ACD and TCMCD shows that, on average, drug-like molecules in MDDR have the highest diversity while natural compounds in TCMCD have the highest complexity. According to the Tree Maps, it can be observed that the Level 1 scaffolds present in MDDR have higher diversity than those presented in TCMCD and ACD. However, some representative scaffolds in MDDR with high frequency show structural similarities to those in TCMCD and ACD, suggesting that some scaffolds in TCMCD and ACD may be potentially drug-like fragments for fragment-based and de novo drug design. PMID:23336706

Scaffolds in Tendon Tissue Engineering

PubMed Central

Longo, Umile Giuseppe; Lamberti, Alfredo; Petrillo, Stefano; Maffulli, Nicola; Denaro, Vincenzo

2012-01-01

Tissue engineering techniques using novel scaffold materials offer potential alternatives for managing tendon disorders. Tissue engineering strategies to improve tendon repair healing include the use of scaffolds, growth factors, cell seeding, or a combination of these approaches. Scaffolds have been the most common strategy investigated to date. Available scaffolds for tendon repair include both biological scaffolds, obtained from mammalian tissues, and synthetic scaffolds, manufactured from chemical compounds. Preliminary studies support the idea that scaffolds can provide an alternative for tendon augmentation with an enormous therapeutic potential. However, available data are lacking to allow definitive conclusion on the use of scaffolds for tendon augmentation. We review the current basic science and clinical understanding in the field of scaffolds and tissue engineering for tendon repair. PMID:22190961

Systematic Prediction of Scaffold Proteins Reveals New Design Principles in Scaffold-Mediated Signal Transduction

PubMed Central

Hu, Jianfei; Neiswinger, Johnathan; Zhang, Jin; Zhu, Heng; Qian, Jiang

2015-01-01

Scaffold proteins play a crucial role in facilitating signal transduction in eukaryotes by bringing together multiple signaling components. In this study, we performed a systematic analysis of scaffold proteins in signal transduction by integrating protein-protein interaction and kinase-substrate relationship networks. We predicted 212 scaffold proteins that are involved in 605 distinct signaling pathways. The computational prediction was validated using a protein microarray-based approach. The predicted scaffold proteins showed several interesting characteristics, as we expected from the functionality of scaffold proteins. We found that the scaffold proteins are likely to interact with each other, which is consistent with previous finding that scaffold proteins tend to form homodimers and heterodimers. Interestingly, a single scaffold protein can be involved in multiple signaling pathways by interacting with other scaffold protein partners. Furthermore, we propose two possible regulatory mechanisms by which the activity of scaffold proteins is coordinated with their associated pathways through phosphorylation process. PMID:26393507

XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes.

PubMed

Saribasak, Huseyin; Maul, Robert W; Cao, Zheng; McClure, Rhonda L; Yang, William; McNeill, Daniel R; Wilson, David M; Gearhart, Patricia J

2011-10-24

Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer's patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1(+/-) splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1(+/-) B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.

Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

PubMed

Yamada, Mari; Watanabe, Yuto; Gootenberg, Jonathan S; Hirano, Hisato; Ran, F Ann; Nakane, Takanori; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2017-03-16

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Induced Förster resonance energy transfer by encapsulation of DNA-scaffold based probes inside a plant virus based protein cage

NASA Astrophysics Data System (ADS)

de Ruiter, Mark V.; Overeem, Nico J.; Singhai, Gaurav; Cornelissen, Jeroen J. L. M.

2018-05-01

Insight into the assembly and disassembly of viruses can play a crucial role in developing cures for viral diseases. Specialized fluorescent probes can benefit the study of interactions within viruses, especially during cell studies. In this work, we developed a strategy based on Förster resonance energy transfer (FRET) to study the assembly of viruses without labeling the exterior of viruses. Instead, we exploit their encapsulation of nucleic cargo, using three different fluorescent ATTO dyes linked to single-stranded DNA oligomers, which are hybridised to a longer DNA strand. FRET is induced upon assembly of the cowpea chlorotic mottle virus, which forms monodisperse icosahedral particles of about 22 nm, thereby increasing the FRET efficiency by a factor of 8. Additionally, encapsulation of the dyes in virus-like particles induces a two-step FRET. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This reversible behavior makes the system a good probe for studying viral assembly and disassembly. It, furthermore, shows that multi-component supramolecular materials are stabilized in the confinement of a protein cage.

Label-free electrochemical genosensor based on mesoporous silica thin film.

PubMed

Saadaoui, Maroua; Fernández, Iñigo; Luna, Gema; Díez, Paula; Campuzano, Susana; Raouafi, Noureddine; Sánchez, Alfredo; Pingarrón, José M; Villalonga, Reynaldo

2016-10-01

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

Scaffolding Student Participation in Mathematical Practices

ERIC Educational Resources Information Center

Moschkovich, Judit N.

2015-01-01

The concept of scaffolding can be used to describe various types of adult guidance, in multiple settings, across different time scales. This article clarifies what we mean by scaffolding, considering several questions specifically for scaffolding in mathematics: What theoretical assumptions are framing scaffolding? What is being scaffolded? At…

Magnesium Oxide Nanoparticles Reinforced Electrospun Alginate-Based Nanofibrous Scaffolds with Improved Physical Properties

PubMed Central

Mantilaka, M. M. M. G. P. G.; Goh, K. L.; Ratnayake, S. P.; Amaratunga, G. A. J.; de Silva, K. M. Nalin

2017-01-01

Mechanically robust alginate-based nanofibrous scaffolds were successfully fabricated by electrospinning method to mimic the natural extracellular matrix structure which benefits development and regeneration of tissues. Alginate-based nanofibres were electrospun from an alginate/poly(vinyl alcohol) (PVA) polyelectrolyte complex. SEM images revealed the spinnability of the complex composite nanofibrous scaffolds, showing randomly oriented, ultrafine, and virtually defects-free alginate-based/MgO nanofibrous scaffolds. Here, it is shown that an alginate/PVA complex scaffold, blended with near-spherical MgO nanoparticles (⌀ 45 nm) at a predetermined concentration (10% (w/w)), is electrospinnable to produce a complex composite nanofibrous scaffold with enhanced mechanical stability. For the comparison purpose, chemically cross-linked electrospun alginate-based scaffolds were also fabricated. Tensile test to rupture revealed the significant differences in the tensile strength and elastic modulus among the alginate scaffolds, alginate/MgO scaffolds, and cross-linked alginate scaffolds (P < 0.05). In contrast to cross-linked alginate scaffolds, alginate/MgO scaffolds yielded the highest tensile strength and elastic modulus while preserving the interfibre porosity of the scaffolds. According to the thermogravimetric analysis, MgO reinforced alginate nanofibrous scaffolds exhibited improved thermal stability. These novel alginate-based/MgO scaffolds are economical and versatile and may be further optimised for use as extracellular matrix substitutes for repair and regeneration of tissues. PMID:28694826

Is scaffold hopping a reliable indicator for the ability of computational methods to identify structurally diverse active compounds?

NASA Astrophysics Data System (ADS)

Dimova, Dilyana; Bajorath, Jürgen

2017-07-01

Computational scaffold hopping aims to identify core structure replacements in active compounds. To evaluate scaffold hopping potential from a principal point of view, regardless of the computational methods that are applied, a global analysis of conventional scaffolds in analog series from compound activity classes was carried out. The majority of analog series was found to contain multiple scaffolds, thus enabling the detection of intra-series scaffold hops among closely related compounds. More than 1000 activity classes were found to contain increasing proportions of multi-scaffold analog series. Thus, using such activity classes for scaffold hopping analysis is likely to overestimate the scaffold hopping (core structure replacement) potential of computational methods, due to an abundance of artificial scaffold hops that are possible within analog series.

Characterization and evaluation of graphene oxide scaffold for periodontal wound healing of class II furcation defects in dog

PubMed Central

Kawamoto, Kohei; Miyaji, Hirofumi; Nishida, Erika; Miyata, Saori; Kato, Akihito; Tateyama, Akito; Furihata, Tomokazu; Shitomi, Kanako; Iwanaga, Toshihiko; Sugaya, Tsutomu

2018-01-01

Introduction The 3-dimensional scaffold plays a key role in volume and quality of repair tissue in periodontal tissue engineering therapy. We fabricated a novel 3D collagen scaffold containing carbon-based 2-dimensional layered material, named graphene oxide (GO). The aim of this study was to characterize and assess GO scaffold for periodontal tissue healing of class II furcation defects in dog. Materials and methods GO scaffolds were prepared by coating the surface of a 3D collagen sponge scaffold with GO dispersion. Scaffolds were characterized using cytotoxicity and tissue reactivity tests. In addition, GO scaffold was implanted into dog class II furcation defects and periodontal healing was investigated at 4 weeks postsurgery. Results GO scaffold exhibited low cytotoxicity and enhanced cellular ingrowth behavior and rat bone forming ability. In addition, GO scaffold stimulated healing of dog class II furcation defects. Periodontal attachment formation, including alveolar bone, periodontal ligament-like tissue, and cementum-like tissue, was significantly increased by GO scaffold implantation, compared with untreated scaffold. Conclusion The results suggest that GO scaffold is biocompatible and possesses excellent bone and periodontal tissue formation ability. Therefore, GO scaffold would be beneficial for periodontal tissue engineering therapy. PMID:29713167

Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue engineering.

PubMed

Huang, Di; Zuo, Yi; Zou, Qin; Wang, Yanying; Gao, Shibo; Wang, Xiaoyan; Liu, Haohuai; Li, Yubao

2012-01-01

High porosity of scaffold is always accompanied by poor mechanical property; the aim of this study was to enhance the strength and modulus of the highly porous scaffold of nanohydroxyapatite/polyamide66 (n-HA/PA66) by coating chitosan (CS) and to investigate the effect of CS content on the scaffold physical properties and cytological properties. The results show that CS coating can reinforce the scaffold effectively. The compress modulus and strength of the CS coated n-HA/PA66 scaffolds are improved to 32.71 and 2.38 MPa, respectively, being about six times and five times of those of the uncoated scaffolds. Meanwhile, the scaffolds still exhibit a highly interconnected porous structure and the porosity is approximate about 78%, slightly lower than the value (84%) of uncoated scaffold. The cytological properties of scaffolds were also studied in vitro by cocultured with osteoblast-like MG63 cells. The cytological experiments demonstrate that the reinforced scaffolds display favorable cytocompatibility and have no significant difference with the uncoated n-HA/PA66 scaffolds. The CS reinforced n-HA/PA66 scaffolds can meet the basic mechanical requirement of bone tissue engineering scaffold, presenting a potential for biomedical application in bone reconstruction and repair. Copyright © 2011 Wiley Periodicals, Inc.

Elastase-Sensitive Elastomeric Scaffolds with Variable Anisotropy for Soft Tissue Engineering

PubMed Central

Guan, Jianjun; Fujimoto, Kazuro L.; Wagner, William R.

2010-01-01

Purpose To develop elastase-sensitive polyurethane scaffolds that would be applicable to the engineering of mechanically active soft tissues. Methods A polyurethane containing an elastase-sensitive peptide sequence was processed into scaffolds by thermally induced phase separation. Processing conditions were manipulated to alter scaffold properties and anisotropy. The scaffold’s mechanical properties, degradation, and cytocompatibility using muscle-derived stem cells were characterized. Scaffold in vivo degradation was evaluated by subcutaneous implantation. Results When heat transfer was multidirectional, scaffolds had randomly oriented pores. Imposition of a heat transfer gradient resulted in oriented pores. Both scaffolds were flexible and relatively strong with mechanical properties dependent upon fabrication conditions such as solvent type, polymer concentration and quenching temperature. Oriented scaffolds exhibited anisotropic mechanical properties with greater tensile strength in the orientation direction. These scaffolds also supported muscle-derived stem cell growth more effectively than random scaffolds. The scaffolds expressed over 40% weight loss after 56 days in elastase containing buffer. Elastase-sensitive scaffolds were complete degraded after 8 weeks subcutaneous implantation in rats, markedly faster than similar polyurethanes that did not contain the peptide sequence. Conclusion The elastase-sensitive polyurethane scaffolds showed promise for application in soft tissue engineering where controlling scaffold mechanical properties and pore architecture are desirable. PMID:18509596

ASDB: a resource for probing protein functions with small molecules.

PubMed

Liu, Zhihong; Ding, Peng; Yan, Xin; Zheng, Minghao; Zhou, Huihao; Xu, Yuehua; Du, Yunfei; Gu, Qiong; Xu, Jun

2016-06-01

: Identifying chemical probes or seeking scaffolds for a specific biological target is important for protein function studies. Therefore, we create the Annotated Scaffold Database (ASDB), a computer-readable and systematic target-annotated scaffold database, to serve such needs. The scaffolds in ASDB were derived from public databases including ChEMBL, DrugBank and TCMSP, with a scaffold-based classification approach. Each scaffold was assigned with an InChIKey as its unique identifier, energy-minimized 3D conformations, and other calculated properties. A scaffold is also associated with drugs, natural products, drug targets and medical indications. The database can be retrieved through text or structure query tools. ASDB collects 333 601 scaffolds, which are associated with 4368 targets. The scaffolds consist of 3032 scaffolds derived from drugs and 5163 scaffolds derived from natural products. For given scaffolds, scaffold-target networks can be generated from the database to demonstrate the relations of scaffolds and targets. ASDB is freely available at http://www.rcdd.org.cn/asdb/with the major web browsers. junxu@biochemomes.com or xujun9@mail.sysu.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A PEGylated platelet free plasma hydrogel based composite scaffold enables stable vascularization and targeted cell delivery for volumetric muscle loss.

PubMed

Aurora, Amit; Wrice, Nicole; Walters, Thomas J; Christy, Robert J; Natesan, Shanmugasundaram

2018-01-01

Extracellular matrix (ECM) scaffolds are being used for the clinical repair of soft tissue injuries. Although improved functional outcomes have been reported, ECM scaffolds show limited tissue specific remodeling response with concomitant deposition of fibrotic tissue. One plausible explanation is the regression of blood vessels which may be limiting the diffusion of oxygen and nutrients across the scaffold. Herein we develop a composite scaffold as a vasculo-inductive platform by integrating PEGylated platelet free plasma (PFP) hydrogel with a muscle derived ECM scaffold (m-ECM). In vitro, adipose derived stem cells (ASCs) seeded onto the composite scaffold differentiated into two distinct morphologies, a tubular network in the hydrogel, and elongated structures along the m-ECM scaffold. The composite scaffold showed a high expression of ITGA5, ITGB1, and FN and a synergistic up-regulation of ang1 and tie-2 transcripts. The in vitro ability of the composite scaffold to provide extracellular milieu for cell adhesion and molecular cues to support vessel formation was investigated in a rodent volumetric muscle loss (VML) model. The composite scaffold delivered with ASCs supported robust and stable vascularization. Additionally, the composite scaffold supported increased localization of ASCs in the defect demonstrating its ability for localized cell delivery. Interestingly, ASCs were observed homing in the injured muscle and around the perivascular space possibly to stabilize the host vasculature. In conclusion, the composite scaffold delivered with ASCs presents a promising approach for scaffold vascularization. The versatile nature of the composite scaffold also makes it easily adaptable for the repair of soft tissue injuries. Decellularized extracellular matrix (ECM) scaffolds when used for soft tissue repair is often accompanied by deposition of fibrotic tissue possibly due to limited scaffold vascularization, which limits the diffusion of oxygen and nutrients across the scaffold. Although a variety of scaffold vascularization strategies has been investigated, their limitations preclude rapid clinical translation. In this study we have developed a composite scaffold by integrating bi-functional polyethylene glycol modified platelet free plasma (PEGylated PFP) with adipose derived stem cells (ASCs) along with a muscle derived ECM scaffold (m-ECM). The composite scaffold provides a vasculo-inductive and an effective cell delivery platform for volumetric muscle loss. Copyright © 2017 Acta Materialia Inc. All rights reserved.

Design, Synthesis, and Evaluation of Novel p-(methylthio)styryl Substituted Quindoline Derivatives as Neuroblastoma RAS (NRAS) Repressors via Specific Stabilizing the RNA G-Quadruplex.

PubMed

Peng, Wang; Sun, Zhi-Yin; Zhang, Qi; Cheng, Sui-Qi; Wang, Shi-Ke; Wang, Xiao-Na; Kuang, Guo-Tao; Su, Xiao-Xuan; Tan, Jia-Heng; Huang, Zhi-Shu; Ou, Tian-Miao

2018-05-25

The human proto-oncogene neuroblastoma RAS (NRAS) contains a guanine-rich sequence in the 5'-untranslated regions (5'-UTR) of the mRNA that could form an RNA G-quadruplex structure. This structure acts as a repressor for NRAS translation and could be a potential target for anti-cancer drugs. Our previous studies found an effective scaffold, the quindoline scaffold, for binding and stabilizing the DNA G-quadruplex structures. Here, basing on the previous studies and reported RNA-specific probes, a series of novel p-(methylthio)styryl substituted quindoline (MSQ) derivatives were designed, synthesized and evaluated as NRAS RNA G-quadruplex ligands. Panels of experiments turned out that the introduction of p-(methylthio)styryl side chain could enhance the specific binding to the NRAS RNA G-quadruplex. One of the hits, 4a-10, showed strong stabilizing activity on the G-quadruplex, and subsequently repressed NRAS's translation and inhibited tumor cells proliferation. Our finding provided a novel strategy to discover novel NRAS repressors by specifically binding to the RNA G-quadruplex in the 5'-UTR of mRNA.

Fission yeast Lem2 and Man1 perform fundamental functions of the animal cell nuclear lamina.

PubMed

Gonzalez, Yanira; Saito, Akira; Sazer, Shelley

2012-01-01

In animal cells the nuclear lamina, which consists of lamins and lamin-associated proteins, serves several functions: it provides a structural scaffold for the nuclear envelope and tethers proteins and heterochromatin to the nuclear periphery. In yeast, proteins and large heterochromatic domains including telomeres are also peripherally localized, but there is no evidence that yeast have lamins or a fibrous nuclear envelope scaffold. Nonetheless, we found that the Lem2 and Man1 proteins of the fission yeast Schizosaccharomyces pombe, evolutionarily distant relatives of the Lap2/Emerin/Man1 (LEM) sub-family of animal cell lamin-associated proteins, perform fundamental functions of the animal cell lamina. These integral inner nuclear membrane localized proteins, with nuclear localized DNA binding Helix-Extension-Helix (HEH) domains, impact nuclear envelope structure and integrity, are essential for the enrichment of telomeres at the nuclear periphery and by means of their HEH domains anchor chromatin, most likely transcriptionally repressed heterochromatin, to the nuclear periphery. These data indicate that the core functions of the nuclear lamina are conserved between fungi and animal cells and can be performed in fission yeast, without lamins or other intermediate filament proteins.

Comparative Biology of Decellularized Lung Matrix: Implications of Species Mismatch in Regenerative Medicine

PubMed Central

Balestrini, Jenna L.; Gard, Ashley L.; Gerhold, Kristin A.; Wilcox, Elise C.; Liu, Angela; Schwan, Jonas; Le, Andrew V.; Baevova, Pavlina; Dimitrievska, Sashka; Zhao, Liping; Sundaram, Sumati; Sun, Huanxing; Rittié, Laure; Dyal, Rachel; Broekelmann, Tom J.; Mecham, Robert P.; Schwartz, Martin A.; Niklason, Laura E.; White, Eric S.

2016-01-01

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration. PMID:27344365

Draft genome sequence of the silver pomfret fish, Pampus argenteus.

PubMed

AlMomin, Sabah; Kumar, Vinod; Al-Amad, Sami; Al-Hussaini, Mohsen; Dashti, Talal; Al-Enezi, Khaznah; Akbar, Abrar

2016-01-01

Silver pomfret, Pampus argenteus, is a fish species from coastal waters. Despite its high commercial value, this edible fish has not been sequenced. Hence, its genetic and genomic studies have been limited. We report the first draft genome sequence of the silver pomfret obtained using a Next Generation Sequencing (NGS) technology. We assembled 38.7 Gb of nucleotides into scaffolds of 350 Mb with N50 of about 1.5 kb, using high quality paired end reads. These scaffolds represent 63.7% of the estimated silver pomfret genome length. The newly sequenced and assembled genome has 11.06% repetitive DNA regions, and this percentage is comparable to that of the tilapia genome. The genome analysis predicted 16 322 genes. About 91% of these genes showed homology with known proteins. Many gene clusters were annotated to protein and fatty-acid metabolism pathways that may be important in the context of the meat texture and immune system developmental processes. The reference genome can pave the way for the identification of many other genomic features that could improve breeding and population-management strategies, and it can also help characterize the genetic diversity of P. argenteus.

Membrane Remodeling by the Double-Barrel Scaffolding Protein of Poxvirus

PubMed Central

Hijnen, Marcel; Schult, Philipp; Pettikiriarachchi, Anne; Mitra, Alok K.; Coulibaly, Fasséli

2011-01-01

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors. PMID:21931553

Effects of a petunia scaffold/matrix attachment region on copy number dependency and stability of transgene expression in Nicotiana tabacum.

PubMed

Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike

2016-04-01

Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.

Structure-guided Design and Immunological Characterization of Immunogens Presenting the HIV-1 gp120 V3 Loop on a CTB Scaffold

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

M Totrov; X Jiang; X Kong

2011-12-31

V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boostingmore » with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.« less

Nanoparticle interface to biology: applications in probing and modulating biological processes.

PubMed

Kah, James Chen Yong; Yeo, Eugenia Li Ling; Koh, Wee Ling; Poinard, Barbara Elodie Ariane; Neo, Dawn Jing Hui

2013-01-01

Nanomaterials can be considered as "pseudo" subcellular entities that are similar to endogenous biomolecules because of their size and ability to interact with other biomolecules. The interaction between nanoparticles and biomolecules gives rise to the nano-bio interface between a nanoparticle and its biological environment. This is often defined in terms of the biomolecules that are present on the surface of the nanoparticles. The nano-bio interface alters the surface characteristics and is what the biological system sees and interacts with. The nanoparticle can thus be viewed as a "scaffold" to which molecules are attached. Intelligent design of this nano-bio interface is therefore crucial to the functionality of nanoscale systems in biology. In this review, we discuss the most common nano-bio interfaces formed from molecules including DNA, polymers, proteins, and antibodies, and discuss their applications in probing and modulating biological processes. We focus our discussion on the nano-bio interface formed on gold nanoparticles as our nanoparticle "scaffold" of interest in part because of our research interest as well as their unique physicochemical properties. While not exhaustive, this review provides a good overview of the latest advances in the use of gold nanomaterial interface to probe and modulate biological processes.

The application of halloysite tubule nanoclay in drug delivery.

PubMed

Lvov, Yuri M; DeVilliers, Melgardt M; Fakhrullin, Rawil F

2016-07-01

Natural and biocompatible clay nanotubes are among the best inorganic materials for drug nanoformulations. These halloysite tubes with SiO2 on the outermost surface have diameter of ca. 50 nm, length around 1 micrometer and may be loaded with drugs at 10-30 wt. %. Narrow tube openings allow for controllable sustained drug release for hours, days or even weeks. Physical-chemical properties of these nanotubes are described followed by examples of drug-loading capabilities, release characteristics, and control of duration of release through the end tube capping with polymers. Development of halloysite-polymer composites such as tissue scaffolds and bone cement/dentist resin formulations with enhanced mechanical properties and extension of the drug release to 2-3 weeks are described. Examples of the compression properties of halloysite in tablets and capsules are also shown. We expect that clay nanotubes will be used primarily for non-injectable drug formulations, such as topical and oral dosage forms, cosmetics, as well as for composite materials with enhanced therapeutic effects. These include tissue scaffolds, bone cement and dentist resins with sustained release of antimicrobial and cell growth-promoting medicines (including proteins and DNA) as well as other formulations such as compounds for antiseptic treatment of hospitals.

Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering.

PubMed

Vikingsson, L; Claessens, B; Gómez-Tejedor, J A; Gallego Ferrer, G; Gómez Ribelles, J L

2015-08-01

In tissue engineering the design and optimization of biodegradable polymeric scaffolds with a 3D-structure is an important field. The porous scaffold provide the cells with an adequate biomechanical environment that allows mechanotransduction signals for cell differentiation and the scaffolds also protect the cells from initial compressive loading. The scaffold have interconnected macro-pores that host the cells and newly formed tissue, while the pore walls should be micro-porous to transport nutrients and waste products. Polycaprolactone (PCL) scaffolds with a double micro- and macro-pore architecture have been proposed for cartilage regeneration. This work explores the influence of the micro-porosity of the pore walls on water permeability and scaffold compliance. A Poly(Vinyl Alcohol) with tailored mechanical properties has been used to simulate the growing cartilage tissue inside the scaffold pores. Unconfined and confined compression tests were performed to characterize both the water permeability and the mechanical response of scaffolds with varying size of micro-porosity while volume fraction of the macro-pores remains constant. The stress relaxation tests show that the stress response of the scaffold/hydrogel construct is a synergic effect determined by the performance of the both components. This is interesting since it suggests that the in vivo outcome of the scaffold is not only dependent upon the material architecture but also the growing tissue inside the scaffold׳s pores. On the other hand, confined compression results show that compliance of the scaffold is mainly controlled by the micro-porosity of the scaffold and less by hydrogel density in the scaffold pores. These conclusions bring together valuable information for customizing the optimal scaffold and to predict the in vivo mechanical behavior. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Mechanical Properties of Tissue Scaffolds by Phase Contrast Imaging and Finite Element Modeling.

PubMed

Bawolin, Nahshon K; Dolovich, Allan T; Chen, Daniel X B; Zhang, Chris W J

2015-08-01

In tissue engineering, the cell and scaffold approach has shown promise as a treatment to regenerate diseased and/or damaged tissue. In this treatment, an artificial construct (scaffold) is seeded with cells, which organize and proliferate into new tissue. The scaffold itself biodegrades with time, leaving behind only newly formed tissue. The degradation qualities of the scaffold are critical during the treatment period, since the change in the mechanical properties of the scaffold with time can influence cell behavior. To observe in time the scaffold's mechanical properties, a straightforward method is to deform the scaffold and then characterize scaffold deflection accordingly. However, experimentally observing the scaffold deflection is challenging. This paper presents a novel study on characterization of mechanical properties of scaffolds by phase contrast imaging and finite element modeling, which specifically includes scaffold fabrication, scaffold imaging, image analysis, and finite elements (FEs) modeling of the scaffold mechanical properties. The innovation of the work rests on the use of in-line phase contrast X-ray imaging at 20 KeV to characterize tissue scaffold deformation caused by ultrasound radiation forces and the use of the Fourier transform to identify movement. Once deformation has been determined experimentally, it is then compared with the predictions given by the forward solution of a finite element model. A consideration of the number of separate loading conditions necessary to uniquely identify the material properties of transversely isotropic and fully orthotropic scaffolds is also presented, along with the use of an FE as a form of regularization.

Chiral plasmonics

PubMed Central

Hentschel, Mario; Schäferling, Martin; Duan, Xiaoyang; Giessen, Harald; Liu, Na

2017-01-01

We present a comprehensive overview of chirality and its optical manifestation in plasmonic nanosystems and nanostructures. We discuss top-down fabricated structures that range from solid metallic nanostructures to groupings of metallic nanoparticles arranged in three dimensions. We also present the large variety of bottom-up synthesized structures. Using DNA, peptides, or other scaffolds, complex nanoparticle arrangements of up to hundreds of individual nanoparticles have been realized. Beyond this static picture, we also give an overview of recent demonstrations of active chiral plasmonic systems, where the chiral optical response can be controlled by an external stimulus. We discuss the prospect of using the unique properties of complex chiral plasmonic systems for enantiomeric sensing schemes. PMID:28560336

Open membranes are the precursors for assembly of large DNA viruses

PubMed Central

Suárez, Cristina; Welsch, Sonja; Chlanda, Petr; Hagen, Wim; Hoppe, Simone; Kolovou, Androniki; Pagnier, Isabelle; Raoult, Didier; Locker, Jacomine Krijnse

2014-01-01

Summary Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convex side of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open-eight’ conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis. PMID:23751082

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.

Fabrication of chitosan/gallic acid 3D microporous scaffold for tissue engineering applications.

PubMed

Thangavel, Ponrasu; Ramachandran, Balaji; Muthuvijayan, Vignesh

2016-05-01

This study explores the potential of gallic acid incorporated chitosan (CS/GA) 3D scaffolds for tissue engineering applications. Scaffolds were prepared by freezing and lyophilization technique and characterized. FTIR spectra confirmed the presence of GA in chitosan (CS) gel. DSC and TGA analysis revealed that the structure of chitosan was not altered due to the incorporation of GA, but thermal stability was significantly increased compared to the CS scaffold. SEM micrographs showed smooth, homogeneous, and microporous architecture of the scaffolds with good interconnectivity. CS/GA scaffolds exhibited approximately 90% porosity on average, increased swelling (600-900%) and controlled biodegradation (15-40%) in PBS (pH 7.4 at 37°C) with 1 mg/mL of lysozyme. CS/GA scaffolds showed 2-4 fold decrease in CFUs (p < 0.05) for both gram positive and gram negative bacteria compared to the CS scaffold. Cytotoxicity of these scaffolds was evaluated using NIH 3T3 L1 fibroblast cells. CS/GA 0.25% scaffold showed similar viability with CS scaffold at 24 and 48 h. CS/GA scaffolds (0.5-1.0%) showed 60-75% viability at 24 h and 90% at 48 h. SEM images showed that an increased cell attachment was observed for CS/GA scaffolds compared to CS scaffolds. These findings authenticate that CS/GA scaffolds were cytocompatible and would be useful for tissue engineering applications. © 2015 Wiley Periodicals, Inc.

Design, construction and mechanical testing of digital 3D anatomical data-based PCL-HA bone tissue engineering scaffold.

PubMed

Yao, Qingqiang; Wei, Bo; Guo, Yang; Jin, Chengzhe; Du, Xiaotao; Yan, Chao; Yan, Junwei; Hu, Wenhao; Xu, Yan; Zhou, Zhi; Wang, Yijin; Wang, Liming

2015-01-01

The study aims to investigate the techniques of design and construction of CT 3D reconstructional data-based polycaprolactone (PCL)-hydroxyapatite (HA) scaffold. Femoral and lumbar spinal specimens of eight male New Zealand white rabbits were performed CT and laser scanning data-based 3D printing scaffold processing using PCL-HA powder. Each group was performed eight scaffolds. The CAD-based 3D printed porous cylindrical stents were 16 piece × 3 groups, including the orthogonal scaffold, the Pozi-hole scaffold and the triangular hole scaffold. The gross forms, fiber scaffold diameters and porosities of the scaffolds were measured, and the mechanical testing was performed towards eight pieces of the three kinds of cylindrical scaffolds, respectively. The loading force, deformation, maximum-affordable pressure and deformation value were recorded. The pore-connection rate of each scaffold was 100 % within each group, there was no significant difference in the gross parameters and micro-structural parameters of each scaffold when compared with the design values (P > 0.05). There was no significant difference in the loading force, deformation and deformation value under the maximum-affordable pressure of the three different cylinder scaffolds when the load was above 320 N. The combination of CT and CAD reverse technology could accomplish the design and manufacturing of complex bone tissue engineering scaffolds, with no significant difference in the impacts of the microstructures towards the physical properties of different porous scaffolds under large load.

Nano-ceramic composite scaffolds for bioreactor-based bone engineering.

PubMed

Lv, Qing; Deng, Meng; Ulery, Bret D; Nair, Lakshmi S; Laurencin, Cato T

2013-08-01

Composites of biodegradable polymers and bioactive ceramics are candidates for tissue-engineered scaffolds that closely match the properties of bone. We previously developed a porous, three-dimensional poly (D,L-lactide-co-glycolide) (PLAGA)/nanohydroxyapatite (n-HA) scaffold as a potential bone tissue engineering matrix suitable for high-aspect ratio vessel (HARV) bioreactor applications. However, the physical and cellular properties of this scaffold are unknown. The present study aims to evaluate the effect of n-HA in modulating PLAGA scaffold properties and human mesenchymal stem cell (HMSC) responses in a HARV bioreactor. By comparing PLAGA/n-HA and PLAGA scaffolds, we asked whether incorporation of n-HA (1) accelerates scaffold degradation and compromises mechanical integrity; (2) promotes HMSC proliferation and differentiation; and (3) enhances HMSC mineralization when cultured in HARV bioreactors. PLAGA/n-HA scaffolds (total number = 48) were loaded into HARV bioreactors for 6 weeks and monitored for mass, molecular weight, mechanical, and morphological changes. HMSCs were seeded on PLAGA/n-HA scaffolds (total number = 38) and cultured in HARV bioreactors for 28 days. Cell migration, proliferation, osteogenic differentiation, and mineralization were characterized at four selected time points. The same amount of PLAGA scaffolds were used as controls. The incorporation of n-HA did not alter the scaffold degradation pattern. PLAGA/n-HA scaffolds maintained their mechanical integrity throughout the 6 weeks in the dynamic culture environment. HMSCs seeded on PLAGA/n-HA scaffolds showed elevated proliferation, expression of osteogenic phenotypic markers, and mineral deposition as compared with cells seeded on PLAGA scaffolds. HMSCs migrated into the scaffold center with nearly uniform cell and extracellular matrix distribution in the scaffold interior. The combination of PLAGA/n-HA scaffolds with HMSCs in HARV bioreactors may allow for the generation of engineered bone tissue. In cases of large bone voids (such as bone cancer), tissue-engineered constructs may provide alternatives to traditional bone grafts by culturing patients' own MSCs with PLAGA/n-HA scaffolds in a HARV culture system.

Cell infiltration and growth in a low density, uncompressed three-dimensional electrospun nanofibrous scaffold.

PubMed

Blakeney, Bryan A; Tambralli, Ajay; Anderson, Joel M; Andukuri, Adinarayana; Lim, Dong-Jin; Dean, Derrick R; Jun, Ho-Wook

2011-02-01

A limiting factor of traditional electrospinning is that the electrospun scaffolds consist entirely of tightly packed nanofiber layers that only provide a superficial porous structure due to the sheet-like assembly process. This unavoidable characteristic hinders cell infiltration and growth throughout the nanofibrous scaffolds. Numerous strategies have been tried to overcome this challenge, including the incorporation of nanoparticles, using larger microfibers, or removing embedded salt or water-soluble fibers to increase porosity. However, these methods still produce sheet-like nanofibrous scaffolds, failing to create a porous three-dimensional scaffold with good structural integrity. Thus, we have developed a three-dimensional cotton ball-like electrospun scaffold that consists of an accumulation of nanofibers in a low density and uncompressed manner. Instead of a traditional flat-plate collector, a grounded spherical dish and an array of needle-like probes were used to create a Focused, Low density, Uncompressed nanoFiber (FLUF) mesh scaffold. Scanning electron microscopy showed that the cotton ball-like scaffold consisted of electrospun nanofibers with a similar diameter but larger pores and less-dense structure compared to the traditional electrospun scaffolds. In addition, laser confocal microscopy demonstrated an open porosity and loosely packed structure throughout the depth of the cotton ball-like scaffold, contrasting the superficially porous and tightly packed structure of the traditional electrospun scaffold. Cells seeded on the cotton ball-like scaffold infiltrated into the scaffold after 7 days of growth, compared to no penetrating growth for the traditional electrospun scaffold. Quantitative analysis showed approximately a 40% higher growth rate for cells on the cotton ball-like scaffold over a 7 day period, possibly due to the increased space for in-growth within the three-dimensional scaffolds. Overall, this method assembles a nanofibrous scaffold that is more advantageous for highly porous interconnectivity and demonstrates great potential for tackling current challenges of electrospun scaffolds. 2010 Elsevier Ltd. All rights reserved.

Pericellular plasma clot negates the influence of scaffold stiffness on chondrogenic differentiation.

PubMed

Arora, Aditya; Kothari, Anjaney; Katti, Dhirendra S

2016-12-01

Matrix stiffness is known to play a pivotal role in cellular differentiation. Studies have shown that soft scaffolds (<2-3kPa) promote cellular aggregation and chondrogenesis, whereas, stiffer ones (>10kPa) show poor chondrogenesis in vitro. In this work we investigated if fibrin matrix from clotted blood can act as a soft surrogate which nullifies the influence of the underlying stiff scaffold, thus promoting chondrogenesis irrespective of bulk scale scaffold stiffness. For this we performed in vitro chondrogenesis on soft (∼1.5kPa) and stiff (∼40kPa) gelatin scaffolds in the presence and absence of pericellular plasma clot. Our results demonstrated that in absence of pericellular plasma clot, chondrocytes showed efficient condensation and cartilaginous matrix secretion only on soft scaffolds, whereas, in presence of pericellular plasma clot, cell rounding and cartilaginous matrix secretion was observed in both soft and stiff scaffolds. More specifically, significantly higher collagen II, chondroitin sulfate and aggrecan deposition was observed in soft scaffolds, and soft and stiff scaffolds with pericellular plasma clot as compared to stiff scaffolds without pericellular plasma clot. Moreover, collagen type I, a fibrocartilage/bone marker was significantly higher only in stiff scaffolds without plasma clot. Therefore, it can be concluded that chondrocytes surrounded by a soft fibrin network were unable to sense the stiffness of the underlying scaffold/substrate and hence facilitate chondrogenesis even on stiff scaffolds. This understanding can have significant implications in the design of scaffolds for cartilage tissue engineering. Cell fate is influenced by the mechanical properties of cell culture substrates. Outside the body, cartilage progenitor cells express significant amounts of cartilage-specific markers on soft scaffolds but not on stiff scaffolds. However, when implanted in joints, stiff scaffolds show equivalent expression of markers as seen in soft scaffolds. This disparity in existing literature prompted our study. Our results suggest that encapsulation of cells in a soft plasma clot, present in any surgical intervention, prevents their perception of stiffness of the underlying scaffold, and hence the ability to distinguish between soft and stiff scaffolds vanishes. This finding would aid the design of new scaffolds that elicit cartilage-like biochemical properties while simultaneously being mechanically comparable to cartilage tissue. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Oxygen Plasma Treatment on 3D-Printed Chitosan/Gelatin/Hydroxyapatite Scaffolds for Bone Tissue Engineering.

PubMed

Lee, Chang-Min; Yang, Seong-Won; Jung, Sang-Chul; Kim, Byung-Hoon

2017-04-01

The 3D hydroxyapatite/gelatin/chitosan composite scaffolds were fabricated by 3D printing technique. The scaffolds were treated by oxygen plasma to improve the bioactivity and its surface characterization and in vitro cell culture were investigated. The scaffolds exhibited the good porosity and interconnectivity of pores. After oxygen plasma etching, roughness and wettability on the scaffolds surface are increased. Plasma treated scaffolds showed higher proliferation than that of untreated scaffolds. Oxygen plasma treatment could be used as potential tool to enhance the biocompatibility on the 3D composite scaffolds.

Modeling and Reconstruction of Micro-structured 3D Chitosan/Gelatin Porous Scaffolds Using Micro-CT

NASA Astrophysics Data System (ADS)

Gong, Haibo; Li, Dichen; He, Jiankang; Liu, Yaxiong; Lian, Qin; Zhao, Jinna

2008-09-01

Three dimensional (3D) channel networks are the key to promise the uniform distribution of nutrients inside 3D hepatic tissue engineering scaffolds and prompt elimination of metabolic products out of the scaffolds. 3D chitosan/gelatin porous scaffolds with predefined internal channels were fabricated and a combination of light microscope, laser confocal microscopy and micro-CT were employed to characterize the structure of porous scaffolds. In order to evaluate the flow field distribution inside the micro-structured 3D scaffolds, a computer reconstructing method based on Micro-CT was proposed. According to this evaluating method, a contrast between 3D porous scaffolds with and without predefined internal channels was also performed to assess scaffolds' fluid characters. Results showed that the internal channel of the 3D scaffolds formed the 3D fluid channel network; the uniformity of flow field distribution of the scaffolds fabricated in this paper was better than the simple porous scaffold without micro-fluid channels.