CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis

Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development. Here, we show by CRISPR/Cas9 techniques that similar to the mouse, neither rb1 nor rbl1 single mosaic mutant Xenopus tropicalis develop tumors, whereas rb1/rbl1 double mosaic mutant tadpoles rapidly develop retinoblastoma. Moreover, occasionally presence of pinealoblastoma (trilateral retinoblastoma) was detected. We thus present the first CRISPR/Cas9 mediated cancer model in Xenopus tropicalis and the first genuine genetic non-mammalian retinoblastoma model. The rapid kinetics of our model paves the way for use as a pre-clinical model. Additionally, this retinoblastoma model provides unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (rb1 + rbl1 + modifier gene) in order to address the clinically unmet need of targeted retinoblastoma therapy

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy

A turbulent model of torque in von Karman swirling flow

A stochastic model is derived to predict the turbulent torque produced by a swirling flow. It is a simple Langevin process, with a colored noise. Using the unified colored noise approximation, we derive analytically the PDF of the fluctuations of injected power in two forcing regimes: constant angular velocity or constant applied torque. In the limit of small velocity fluctuations and vanishing inertia, we predict that the injected power fluctuates twice less in the case of constant torque than in the case of constant angular velocity forcing. The model is further tested against experimental data in a von Karman device filled with water. It is shown to allow for a parameter-free prediction of the PDF of power fluctuations in the case where the forcing is made at constant torque. A physical interpretation of our model is finally given, using a quasi-linear model of turbulence

Entropy production and Lyapunov instability at the onset of turbulent convection

Computer simulations of a compressible fluid, convecting heat in two dimensions, suggest that, within a range of Rayleigh numbers, two distinctly different, but stable, time-dependent flow morphologies are possible. The simpler of the flows has two characteristic frequencies: the rotation frequency of the convecting rolls, and the vertical oscillation frequency of the rolls. Observables, such as the heat flux, have a simple-periodic (harmonic) time dependence. The more complex flow has at least one additional characteristic frequency -- the horizontal frequency of the cold, downward- and the warm, upward-flowing plumes. Observables of this latter flow have a broadband frequency distribution. The two flow morphologies, at the same Rayleigh number, have different rates of entropy production and different Lyapunov exponents. The simpler "harmonic" flow transports more heat (produces entropy at a greater rate), whereas the more complex "chaotic" flow has a larger maximum Lyapunov exponent (corresponding to a larger rate of phase-space information loss). A linear combination of these two rates is invariant for the two flow morphologies over the entire range of Rayleigh numbers for which the flows coexist, suggesting a relation between the two rates near the onset of convective turbulence.Comment: 5 pages, 4 figure

Bone Tissue Response to Porous and Functionalized Titanium and Silica Based Coatings

Background: Topography and presence of bio-mimetic coatings are known to improve osseointegration. The objective of this study was to evaluate the bone regeneration potential of porous and osteogenic coatings. Methodology: Six-implants [Control (CTR); porous titanium coatings (T1, T2); thickened titanium (Ti) dioxide layer (TiO2); Amorphous Microporous Silica (AMS) and Bio-active Glass (BAG)] were implanted randomly in tibiae of 20-New Zealand white rabbits. The animals were sacrificed after 2 or 4 weeks. The samples were analyzed histologically and histomorphometrically. In the initial bone-free areas (bone regeneration areas (BRAs)), the bone area fraction (BAF) was evaluated in the whole cavity (500 mm, BAF-500), in the implant vicinity (100 mm, BAF-100) and further away (100–500 mm, BAF-400) from the implant. Bone-to-implant contact (BIC-BAA) was measured in the areas where the implants were installed in contact to the host bone (bone adaptation areas (BAAs)) to understand and compare the bone adaptation. Mixed models were used for statistical analysis. Principal Findings: After 2 weeks, the differences in BAF-500 for different surfaces were not significant (p.0.05). After 4 weeks, a higher BAF-500 was observed for BAG than CTR. BAF-100 for AMS was higher than BAG and BAF-400 for BAG was higher than CTR and AMS. For T1 and AMS, the bone regeneration was faster in the 100-mm compared to the 400-mm zone. BIC-BAA for AMS and BAG was lower after 4 than 2 weeks. After 4 weeks, BIC-BAA for BAG was lower than AMS and CTR. Conclusions: BAG is highly osteogenic at a distance from the implant. The porous titanium coatings didn’t stimulate bone regeneration but allowed bone growth into the pores. Although AMS didn’t stimulate higher bone response, it has a potential of faster bone growth in the vicinity compared to further away from the surface. BIC-BAA data were inconclusive to understand the bone adaptation.status: publishe