Closed hybrid hierarchical reservoirs based on the deconstruction of the cellular native microenvironment

The stem cell niche organization and dynamics provide valuable cues for the development of mimetic environments that could have potential to stimulate the regenerative process. We propose the use of biodegradable biomaterials to produce closed miniaturised structures able to encapsulate different cell types or bioactive molecules. In particular, capsules are fabricated using the so-called layer-by-layer technology, where the consecutive (nano-sized) layers are well stabilized by electrostatic interactions or other weak forces. Using alginate-based spherical templates containing cells or other elements (e.g. proteins, magnetic nanoparticles, microparticles) it is possible to produce liquefied capsules that may entrap the entire cargo under mild conditions. The inclusion of liquefied micropcapsules may be used to produce hierarchical compartmentalised systems for the delivery of bioactive agents. The presence of solid microparticles inside such capsules offers adequate surface area for adherent cell attachment increasing the biological performance of these hierarchical systems, while maintain both permeability and injectability. We demonstrated that the encapsulation of distinct cell types (including mesenchymal stem cells and endothelial cells) enhances the osteogenic capability of this system, that could be useful in bone tissue engineering applications.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia Type 3 pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.