Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers

Background: Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful nucleic acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key step is a pre-requisite for performing LNA-modified RNA aptamer selection

Nanostructured 3D Constructs Based on Chitosan and Chondroitin Sulphate Multilayers for Cartilage Tissue Engineering

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

Secondary structure of rhBMP-2 in a protective biopolymeric carrier material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.This work was supported by the French Ministry of Research through an ANR-EmergenceBIO grant (ANR-09-EBIO-012-01), by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370), and by GRAVIT (081012_FIBIOS). C.P. is grafetul to IUF for financial support

Free-standing polyelectrolyte membranes made of chitosan and alginate

Free-standing films have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we prepared free-standing membranes by the layer-by-layer assembly of chitosan and alginate, two widely used biomaterials. Our aim was to produce a thick membrane and to study the permeation of model drugs and the adhesion of muscle cells. We first defined the optimal growth conditions in terms of pH and alginate concentration. The membranes could be easily detached from polystyrene or polypropylene substrate without any postprocessing step. The dry thickness was varied over a large range from 4 to 35 μm. A 2-fold swelling was observed by confocal microscopy when they were immersed in PBS. In addition, we quantified the permeation of model drugs (fluorescent dextrans) through the free-standing membrane, which depended on the dextran molecular weight. Finally, we showed that myoblast cells exhibited a preferential adhesion on the alginate-ending membrane as compared to the chitosan-ending membrane or to the substrate side.This work was financially supported by Foundation for Science and Technology (FCT) through the Scholarship SFRH/BD/64601/2009 granted to S.G.C. C.M. is indebted to Grenoble INP for financial support via a postdoctoral fellowship. This work was supported by the European Commission (FP7 Program) via a European Research Council starting grant (BIOMIM, GA 259370 to C.P.). C.P. is also grateful to Institut Universitaire de France and to Grenoble Institute of Technology for financial support. We thank Isabelle Paintrand for her technical help with the confocal apparatus and Patrick Chaudouet for his help with SEM imaging

Morphological, behavioral and cellular analyses revealed different phenotypes in Wolfram syndrome wfs1a and wfs1b zebrafish mutant lines

Wolfram syndrome (WS) is a rare genetic disease characterized by diabetes, optic atrophy and deafness. Patients die at 35 years of age, mainly from respiratory failure or dysphagia. Unfortunately, there is no treatment to block the progression of symptoms and there is an urgent need for adequate research models. Here, we report on the phenotypical characterization of two loss-of-function zebrafish mutant lines: wfs1aC825X and wfs1bW493X. We observed that wfs1a deficiency altered the size of the ear and the retina of the fish. We also documented a decrease in the expression level of unfolded protein response (UPR) genes in basal condition and in stress condition, i.e. after tunicamycin treatment. Interestingly, both mutants lead to a decrease in their visual function measured behaviorally. These deficits were associated with a decrease in the expression level of UPR genes in basal and stress conditions. Interestingly, basal, ATP-linked and maximal mitochondrial respirations were transiently decreased in the wfs1b mutant. Taken together, these zebrafish lines highlight the critical role of wfs1a and wfs1b in UPR, mitochondrial function and visual physiology. These models will be useful tools to better understand the cellular function of Wfs1 and to develop novel therapeutic approaches for WS

Bacteria-responsive multilayer coatings comprising polycationic nanospheres for bacteria biofilm prevention on urinary catheters

This work reports on the development of infection-preventive coatings on silicone urinary catheters that contain in their structure and release on demand antibacterial polycationic nanospheres. Polycationic aminocellulose conjugate was first sonochemically processed into nanospheres to improve its antibacterial potential compared to the bulk conjugate in solution (ACSol). Afterwards the processed aminocellulose nanospheres (ACNSs) were combined with the hyaluronic acid (HA) polyanion to build a layer-by-layer construct on silicone surfaces. Although the coating deposition was more effective when HA was coupled with ACSol than with ACNSs, the ACNSs-based coatings were thicker and displayed smoother surfaces due to the embedment of intact nanospheres. The antibacterial effect of the ACNSs multilayers was by 40 % higher compared to the ACSol coatings. This fact was further translated into more effective prevention of Pseudomonas aeruginosa biofilm formation. The coatings were stable in absence of bacteria, whereas their disassembling occurred gradually during incubation with Pseudomonas aeruginosa, and thus eradicate the biofilm upon release of antibacterial agents. Only 5 bilayers of HA/ACNSs were sufficient to prevent the biofilm formation, in contrast to the 10 bilayers of ACSol required to achieve the same effect. The antibiofilm efficiency of (HA/ACNSs)10 multilayer construct built on a Foley catheter was additionally validated under dynamic conditions using a model of catheterized bladder in which the biofilm was grown during seven days.M.M.F. acknowledges the support of the European Commissionunder the Marie Curie Intra-European Fellowship (IEF) Program (Grant Agreement ‘‘NanoQuench” FP7-331416)

Towards the design of 3D multiscale instructive tissue engineering constructs: Current approaches and trends

The design of 3D constructs with adequate properties to instruct and guide cells both in vitro and in vivo is one of the major focuses of tissue engineering. Successful tissue regeneration depends on the favorable crosstalk between the supporting structure, the cells and the host tissue so that a balanced matrix production and degradation is achieved. Herein, the major occurring events and players in normal and regenerative tissue are overviewed. These have been inspiring the selection or synthesis of instructive cues to include into the 3D constructs. We further highlight the importance of a multiscale perception of the range of features that can be included on the biomimetic structures. Lastly, we focus on the current and developing tissue-engineering approaches for the preparation of such 3D constructs: top-down, bottom-up and integrative. Bottom-up and integrative approaches present a higher potential for the design of tissue engineering devices with multiscale features and higher biochemichal control than top-down strategies, and are the main focus of this review.The research leading to these results has received funding from the European Research Council grant agreement ERC-2012-ADG-20120216-321266 for the project ComplexiTE. Portuguese Foundation for Science and Technology is gratefully acknowledged for the fellowship of Sara M. Oliveira (SFRH/BD/70107/2010)

Faint objects in motion: the new frontier of high precision astrometry

Funder: Istituto Nazionale di AstrofisicaSky survey telescopes and powerful targeted telescopes play complementary roles in astronomy. In order to investigate the nature and characteristics of the motions of very faint objects, a flexibly-pointed instrument capable of high astrometric accuracy is an ideal complement to current astrometric surveys and a unique tool for precision astrophysics. Such a space-based mission will push the frontier of precision astrometry from evidence of Earth-mass habitable worlds around the nearest stars, to distant Milky Way objects, and out to the Local Group of galaxies. As we enter the era of the James Webb Space Telescope and the new ground-based, adaptive-optics-enabled giant telescopes, by obtaining these high precision measurements on key objects that Gaia could not reach, a mission that focuses on high precision astrometry science can consolidate our theoretical understanding of the local Universe, enable extrapolation of physical processes to remote redshifts, and derive a much more consistent picture of cosmological evolution and the likely fate of our cosmos. Already several missions have been proposed to address the science case of faint objects in motion using high precision astrometry missions: NEAT proposed for the ESA M3 opportunity, micro-NEAT for the S1 opportunity, and Theia for the M4 and M5 opportunities. Additional new mission configurations adapted with technological innovations could be envisioned to pursue accurate measurements of these extremely small motions. The goal of this White Paper is to address the fundamental science questions that are at stake when we focus on the motions of faint sky objects and to briefly review instrumentation and mission profiles