Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors

Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. However, it will require further investigation to determine whether the degree of binding and tight association between DNA and nanotubes is a desirable trait to increase gene expression efficiency in vitro or in vivo. This study constitutes the first thorough investigation into the physicochemical interactions between cationic functionalized carbon nanotubes and DNA toward construction of carbon nanotube-based gene transfer vector systems.     

TRIPLET YIELD OF MEROCYANINE 540 IN WATER IS WAVELENGTH DEPENDENT

Monomers and aggregates of Merocyanine 540 (MC540) in water are able to photoisomerize. The shape of the photoisomer absorption spectrum is very similar to that of the ground state. Triplet state of MC540 in water has been produced by energy transfer from triplet anthracene and displays a broad absorption spectrum between 600 and 700 nm. The triplet state may also be produced by direct excitation of MC540 with UV light. However, when the dye is excited by visible light, no triplet state absorbance in the red could be detected so that the triplet yield of MC540 in water seems to be excitation wavelength dependent.     

Network virtualization as an integrated solution for emergency communication

In this paper the Virtual Private Ad Hoc Networking (VPAN) platform is introduced as an integrated networking solution for many applications that require secure transparent continuous connectivity using heterogeneous devices and network technologies. This is done by creating a virtual logical self-organizing network on top of existing network technologies reducing complexity and maintaining session continuity right from the start. One of the most interesting applications relies in the field of emergency communication with its specific needs which will be discussed in this paper and matched in detail against the architecture and features of the VPAN platform. The concept and dynamics are demonstrated and evaluated with measurements done on real hardware.     

Virtual Private Ad Hoc Networking

The fact that a lot of applications require secure communication to take place only between a dynamic subset of distributed devices sharing a common context, is, from a network point of view, very challenging and demanding. Existing technologies such as VPN, P2P overlays or VLANs can only partially respond to these requirements. This observation is the key factor that has driven the proposal of the virtual private ad hoc network concept. Virtual private ad hoc networks (VPAN) are secure and self-organizing overlay networks on top of existing IP infrastructure that use ad hoc networking techniques to enable network connectivity. The underlying IP infrastructure can be the Internet, cellular networks, ad hoc networks, mesh networks … or combinations thereof. A virtual private ad hoc overlay network creates a transparent, shielded and trusted environment for the applications and services running on the participants' devices. The overlay uses internal addressing and ad hoc routing, thereby forming a virtual network on top of the physical infrastructure. In addition, the overlay must be self-organizing and self-maintaining upon member mobility or membership changes. This paper gives an overview of the potential applications, a high-level network architecture and the network challenges emerging from the novel concept of virtual private ad hoc networking. Jeroen Hoebeke was born in Ghent, Belgium in 1979. In 2002 he received the Masters degree in engineering (Computer Science) from the University of Ghent. In August 2002, he joined the Broadband Communications Networks Group. His PhD research includes the development of adaptive routing protocol techniques for mobile ad hoc networks. His main research interests are in ad hoc wireless communications and, more generally, in broadband wireless communications. Within the European MAGNET project, he is actively involved in the development of a network architecture and demonstrator for Personal Networks, with a prime focus on routing and connectivity. Gerry Holderbeke was born in Zottegem, Belgium in 1982. He graduated in Informatics at the University of Ghent in 2004. In August 2004 he joined the Broadband Communications Networks Group where he is currently working as a project developer. His research currently includes the development of an emulator for mobile ad hoc networks. His main research interests are in ad hoc networks and broadband wireless communications and involve routing, addressing and more generally, communication within mobile ad hoc networks and infrastructured networks. Within the European MAGNET project, he is actively involved in the development of a network architecture for Personal Networks, with a prime focus on the implementation of the routing architecture. Ingrid Moerman was born in Gent, Belgium in 1965. She received the degree in Electro-technical Engineering and the Ph.D degree from the Ghent University, Gent, Belgium in 1987 and 1992, respectively. Since 1987, she has been with the Interuniversity Micro-Electronics Centre (IMEC) at the Department of Information Technology (INTEC) of the Ghent University, where she conducted research in the field of optoelectronics. In 1997, she became a permanent member of the Research Staff at IMEC. Since 2000 she is part-time professor at the Ghent University. Since 2001 she has switched her research domain to broadband communication networks. She is currently involved in the research and education on broadband mobile & wireless communication networks and on multimedia over IP. The main research topics related to mobile & wireless communication networks are: wireless access to vehicles (high bandwidth & driving speed), adaptive QoS routing in wireless ad hoc networks, body area networks, protocol boosting on wireless links, design of fixed access/metro part, traffic engineering and QoS support in the wireless access network. Ingrid Moerman is author or co-author of more than 300 publications in the field of optoelectronics and communication networks. Bart Dhoedt received a degree in Engineering from the Ghent University in 1990. In September 1990, he joined the Department of Information Technology of the Faculty of Applied Sciences, University of Ghent. His research, addressing the use of micro-optics to realize parallel free space optical interconnects, resulted in a PhD degree in 1995. After a 2 year post-doc in opto-electronics, he became professor at the Faculty of Applied Sciences, Department of Information Technology. Since then, he is responsible for several courses on algorithms, programming and software development. His research interests are software engineering and mobile & wireless communications. Bart Dhoedt is author or co-author of approximately 70 papers published in international journals or in the proceedings of international conferences. His current research addresses software technologies for communication networks, peer-to-peer networks, mobile networks and active networks. Piet Demeester received the Masters degree in Electro-technical engineering and the Ph.D degree from the Ghent University, Gent, Belgium in 1984 and 1988, respectively. In 1992 he started a new research activity on broadband communication networks resulting in the IBCN-group (INTEC Broadband communications network research group). Since 1993 he became professor at the Ghent University where he is responsible for the research and education on communication networks. The research activities cover various communication networks (IP, ATM, SDH, WDM, access, active, mobile), including network planning, network and service management, telecom software, internetworking, network protocols for QoS support, etc. Piet Demeester is author of more than 300 publications in the area of network design, optimization and management. He is member of the editorial board of several international journals and has been member of several technical program committees (ECOC, OFC, DRCN, ICCCN, IZS, &).     

Solid-phase synthesis and characterization of a novel fullerene-peptide derived from histone H3

A peptide analogue from a histone H3 protein containing the L-fulleropyrrolidino-glutamic acid has been prepared by a solid-phase approach and has been fully characterized. By molecular modelling it was verified that this peptide derivative is able to retain a binding capacity to the MHC (major histocompatibility complex) molecule similar to that of the cognate epitope.     

Disentangling the Information in Species Interaction Networks

Shannon’s entropy measure is a popular means for quantifying ecological diversity. We explore how one can use information-theoretic measures (that are often called indices in ecology) on joint ensembles to study the diversity of species interaction networks. We leverage the little-known balance equation to decompose the network information into three components describing the species abundance, specificity, and redundancy. This balance reveals that there exists a fundamental trade-off between these components. The decomposition can be straightforwardly extended to analyse networks through time as well as space, leading to the corresponding notions for alpha, beta, and gamma diversity. Our work aims to provide an accessible introduction for ecologists. To this end, we illustrate the interpretation of the components on numerous real networks. The corresponding code is made available to the community in the specialised Julia package EcologicalNetworks.jl.     

Electron Spin Resonance Evidence of the Generation of Superoxide Anion, Hydroxyl Radical and Singlet Oxygen during the Photohemolysis of Human Erythrocytes with Bacteriochlorin a

Abstract— Photodynamic therapy with bacteriochlorin a (BCA) as sensitizer induces damage to red blood cells in vivo. To assess the extent of the contributuion of reactive oxygen species (ROS) and to determine a possible reaction mechanism, competition experiments with assorted ROS quenching or/and enhancing agents were performed in human erythrocytes as model system and in phosphate buffer. In the erythrocyte experiments, a 2% suspension was incubated with BCA for 1 h, washed with phosphate-buffered saline, resuspended and subsequently illuminated with a diode laser using a fluence rate of 2.65 mW/cm₂. Potassium leakage and hemolysis were light and BCA dose dependent. Adding tryptophan (3.3 mM), azide (1 mM) or histidine (10 mM) to the erythrocyte suspension before illumination delayed the onset of K-leakage and hemolysis suggesting a type II mechanism. The D₂O did not affect K-leakage nor photohemolysis. Adding mannitol (13.3 mM) or glycerol (300 nM) also caused a delay in the onset of K-leakage and hemolysis, suggesting the involvement of radicals. In phosphate buffer experiments, it was shown using electron spin resonance (ESR) associated with spin-trapping techniques that BCA is able to generate 0₂～* and OH* radicals without production of aqueous electron. Visible or UV irradiation of the dye in the presence of the spin trap 5,5-dimethyl-1-pyrroline-iV-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct DMPO-OH. Addition of ethanol or sodium formate produced supplementary hyperfine splittings due to the respective CH₃CHOH _* and CO_2-1 radical adducts, indicating the presence of free OH_*. Production of DMPO-OH was partly inhibited by superoxide dismutase (SOD), catalase and desferoxamine, suggesting that the iron-catalyzed decomposition of H₂O₂ was partly involved in the formation of one part of the observed OH _*. The complementary inhibition of DMPO-OH production by azide and 9,10-anthracenedipropionic acid (ADPA) was consistent with ₁O₂ production by BCA followed by reaction of ₁O₂ with DMPO and decay of the intermediate complex to form DMPO-OH and free OH_*. All our results seem to indicate that BCA is a 50%/50% type 1/type 2 sensitizer in buffered aqueous solutions and confirmed that the dye-induced hemolysis of erythrocytes was well caused by a mixed type 1/type 2 mechanism.     

Bringing Time-Sensitive Networking to Wireless Professional Private Networks

Wireless Personal Communications - Private professional environments such as manufacturing industry, warehouses, hospitals, airports, among others, increasingly rely on end-to-end connected...     

Small multivalent architectures mimicking homotrimers of the TNF superfamily member CD40L: delineating the relationship between structure and effector function

Synthetic multivalent ligands, owing to the presence of multiple copies of a recognition motif attached to a central scaffold, can mediate clustering of cell surface receptors and thereby function as effector molecules. This paper dissects the relationship between structure and effector function of synthetic multivalent ligands targeting CD40, a cell surface receptor of the tumor necrosis factor receptor (TNF-R) superfamily. Triggering CD40 signaling in vivo can be used to enhance immunity against intracellular pathogens or tumors. A series of multimeric molecules has been prepared by systematically varying the shape and the valency of the central scaffold, the nature and the length of the linker as well as the sequence of the receptor binding motif. The data reported here (i) suggest that radial distribution of CD40-binding units and C3-symmetry are preferred for optimal binding to CD40 and signaling, (ii) underscore the importance of choosing an appropriate linker to connect the receptor binding motif to the central scaffold, and (iii) show the versatility of planar cyclic alpha- and beta-peptides as templates for the design of CD40L mimetics. In particular, the (Ahx)3-B trimeric scaffold-linker combination equally accommodated binding elements derived from distinct CD40L hot-spot regions including AA" loop and beta-strand E. The use of miniCD40Ls such as those reported here is complementary to other approaches (recombinant ligands, agonistic anti-receptor antibodies) and may find interesting therapeutic applications. Furthermore, the results disclosed in this paper provide the basis for future design of other TNF family member mimetics.     

Characterisation of antibodies and analytes by surface plasmon resonance for the optimisation of a competitive immunoassay based on energy transfer

The determination of binding constants using surface plasmon resonance (SPR) was introduced to optimise a competitive homogeneous fluorescence energy-transfer immunoassay (ETIA) before labelling. Steroids were chosen as model for the detection of three analytes estrone, estradiol and ethinylestradiol--by taking three polyclonal antibodies (anti estrone-, anti estradiol- and anti estrogen-antibodies) and the corresponding analyte derivatives used for the immunisation. The active concentration of the antibodies was determined before and after labelling. Inhibition curves were recorded using SPR for all possible combinations of analyte, antibody, and analyte derivatives. The experiments revealed that the active antibody concentration can be reduced to 30% whereas the antibody affinity is not affected by the labelling process. Limits of the use of SPR for determination of affinity constants in solution are discussed. All possible ETIA calibration for the quantification of estrone and estradiol was performed. The lower limits of detection for estrone (0.06 microg L(-1)) and estradiol (0.17 microg L(-1)) were reached with the anti-estrogen IgG and its derivative