Improved osteogenic vector for non-viral gene therapy

Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches

Constitutive and inducible co-expression systems for non-viral osteoinductive gene therapy

Tissue regenerative gene therapy requires expression strategies that deliver therapeutic effective amounts of transgenes. As physiological expression patterns are more complex than high-level expression of a singular therapeutic gene, we aimed at constitutive or inducible co-expression of 2 transgenes simultaneously. Co-expression of human bone morphogenetic protein 2 and 7 (BMP2/7) from constitutively expressing and doxycycline inducible plasmids was evaluated in vitro in C2C12 cells with osteocalcin reporter gene assays and standard assays for osteogenic differentiation. The constitutive systems were additionally tested in an in vivo pilot for ectopic bone formation after repeated naked DNA injection to murine muscle tissue. Inductor controlled differentiation was demonstrated in vitro for inducible co-expression. Both co-expression systems, inducible and constitutive, achieved significantly better osteogenic differentiation than single factor expression. The potency of the constitutive co-expression systems was dependent on relative expression cassette topology. In vivo, ectopic bone formation was demonstrated in 6/13 animals (46 % bone formation efficacy) at days 14 and 28 in hind limb muscles as proven by in vivo μCT and histological evaluation. In vitro findings demonstrated that the devised single vector BMP2/7 co-expression strategy mediates superior osteoinduction, can be applied in an inductor controlled fashion and that its efficiency is dependent on expression cassette topology. In vivo results indicate that co-expression of BMP2/7 applied by non-viral naked DNA gene transfer effectively mediates bone formation without the application of biomaterials, cells or recombinant growth factors, offering a promising alternative to current treatment strategies with potential for clinical translation in the future

Size Dependence of Metal-Insulator Transition in Stoichiometric Fe3O4 Nanocrystals

Magnetite (Fe3O4) is one of the most actively studied materials with a famous metal-insulator transition (MIT), so-called the Verwey transition at around 123 K. Despite the recent progress in synthesis and characterization of Fe3O4 nanocrystals (NCs), it is still an open question how the Verwey transition changes on a nanometer scale. We herein report the systematic studies on size dependence of the Verwey transition of stoichiometric Fe3O4 NCs. We have successfully synthesized stoichiometric and uniform-sized Fe3O4 NCs with sizes ranging from 5 to 100 nm. These stoichiometric Fe3O4 NCs show the Verwey transition when they are characterized by conductance, magnetization, cryo-XRD, and heat capacity measurements. The Verwey transition is weakly size-dependent and becomes suppressed in NCs smaller than 20 nm before disappearing completely for less than 6 nm, which is a clear, yet highly interesting indication of a size effect of this well-known phenomena. Our current work will shed new light on this ages-old problem of Verwey transition.Comment: 18 pages, 4 figures, Nano Letters (accepted

Limits on the high-energy gamma and neutrino fluxes from the SGR 1806-20 giant flare of December 27th, 2004 with the AMANDA-II detector

On December 27th 2004, a giant gamma flare from the Soft Gamma-ray Repeater 1806-20 saturated many satellite gamma-ray detectors. This event was by more than two orders of magnitude the brightest cosmic transient ever observed. If the gamma emission extends up to TeV energies with a hard power law energy spectrum, photo-produced muons could be observed in surface and underground arrays. Moreover, high-energy neutrinos could have been produced during the SGR giant flare if there were substantial baryonic outflow from the magnetar. These high-energy neutrinos would have also produced muons in an underground array. AMANDA-II was used to search for downgoing muons indicative of high-energy gammas and/or neutrinos. The data revealed no significant signal. The upper limit on the gamma flux at 90% CL is dN/dE < 0.05 (0.5) TeV^-1 m^-2 s^-1 for gamma=-1.47 (-2). Similarly, we set limits on the normalization constant of the high-energy neutrino emission of 0.4 (6.1) TeV^-1 m^-2 s^-1 for gamma=-1.47 (-2).Comment: 14 pages, 3 figure

Observation of High-Energy Astrophysical Neutrinos in Three Years of IceCube Data

A search for high-energy neutrinos interacting within the IceCube detector between 2010 and 2012 provided the first evidence for a high-energy neutrino flux of extraterrestrial origin. Results from an analysis using the same methods with a third year (2012-2013) of data from the complete IceCube detector are consistent with the previously reported astrophysical flux in the 100 TeV - PeV range at the level of $10^{-8}\, \mathrm{GeV}\, \mathrm{cm}^{-2}\, \mathrm{s}^{-1}\, \mathrm{sr}^{-1}$ per flavor and reject a purely atmospheric explanation for the combined 3-year data at $5.7 \sigma$. The data are consistent with expectations for equal fluxes of all three neutrino flavors and with isotropic arrival directions, suggesting either numerous or spatially extended sources. The three-year dataset, with a livetime of 988 days, contains a total of 37 neutrino candidate events with deposited energies ranging from 30 to 2000 TeV. The 2000 TeV event is the highest-energy neutrino interaction ever observed.Comment: 8 pages, 5 figures. Accepted by PRL. The event catalog, event displays, and other data tables are included after the final page of the article. Changed from the initial submission to reflect referee comments, expanding the section on atmospheric backgrounds, and fixes offsets of up to 0.9 seconds in reported event times. Address correspondence to: J. Feintzeig, C. Kopper, N. Whitehor

Atmospheric and Astrophysical Neutrinos above 1 TeV Interacting in IceCube

The IceCube Neutrino Observatory was designed primarily to search for high-energy (TeV--PeV) neutrinos produced in distant astrophysical objects. A search for $\gtrsim 100$~TeV neutrinos interacting inside the instrumented volume has recently provided evidence for an isotropic flux of such neutrinos. At lower energies, IceCube collects large numbers of neutrinos from the weak decays of mesons in cosmic-ray air showers. Here we present the results of a search for neutrino interactions inside IceCube's instrumented volume between 1~TeV and 1~PeV in 641 days of data taken from 2010--2012, lowering the energy threshold for neutrinos from the southern sky below 10 TeV for the first time, far below the threshold of the previous high-energy analysis. Astrophysical neutrinos remain the dominant component in the southern sky down to 10 TeV. From these data we derive new constraints on the diffuse astrophysical neutrino spectrum, $\Phi_{\nu} = 2.06^{+0.4}_{-0.3} \times 10^{-18} \left({E_{\nu}}/{10^5 \,\, \rm{GeV}} \right)^{-2.46 \pm 0.12} {\rm {GeV^{-1} \, cm^{-2} \, sr^{-1} \, s^{-1}} }$, as well as the strongest upper limit yet on the flux of neutrinos from charmed-meson decay in the atmosphere, 1.52 times the benchmark theoretical prediction used in previous IceCube results at 90\% confidence.Comment: 18 pages, 12 figure

Search for Prompt Neutrino Emission from Gamma-Ray Bursts with IceCube

We present constraints derived from a search of four years of IceCube data for a prompt neutrino flux from gamma-ray bursts (GRBs). A single low-significance neutrino, compatible with the atmospheric neutrino background, was found in coincidence with one of the 506 observed bursts. Although GRBs have been proposed as candidate sources for ultra-high energy cosmic rays, our limits on the neutrino flux disfavor much of the parameter space for the latest models. We also find that no more than $\sim1\%$ of the recently observed astrophysical neutrino flux consists of prompt emission from GRBs that are potentially observable by existing satellites.Comment: 15 pages, 3 figure

Search for a diffuse flux of astrophysical muon neutrinos with the IceCube 59-string configuration

A search for high-energy neutrinos was performed using data collected by the IceCube Neutrino Observatory from May 2009 to May 2010, when the array was running in its 59-string configuration. The data sample was optimized to contain muon neutrino induced events with a background contamination of atmospheric muons of less than 1%. These data, which are dominated by atmospheric neutrinos, are analyzed with a global likelihood fit to search for possible contributions of prompt atmospheric and astrophysical neutrinos, neither of which have yet been identified. Such signals are expected to follow a harder energy spectrum than conventional atmospheric neutrinos. In addition, the zenith angle distribution differs for astrophysical and atmospheric signals. A global fit of the reconstructed energies and directions of observed events is performed, including possible neutrino flux contributions for an astrophysical signal and atmospheric backgrounds as well as systematic uncertainties of the experiment and theoretical predictions. The best fit yields an astrophysical signal flux for nu(mu) + (nu) over bar (mu) of E-2. Phi(E) = 0.25 x 10(-8) GeV cm(-2) s(-1) sr(-1), and a zero prompt component. Although the sensitivity of this analysis for astrophysical neutrinos surpasses the Waxman and Bahcall upper bound, the experimental limit at 90% confidence level is a factor of 1.5 above at a flux of E-2 . Phi(E) = 1.44 x 10(-8) GeV cm(-2) s(-1) sr(-1)