Identification and characterization of a novel potential transcription factor involved in osteogenic differentiation of mesenchymal stem cells.

Osteogenesis is a complex and still poorly understood biological process regulated by intrinsic cellular signals and extrinsic micro-environmental stimuli from the surrounding stem cell-niche. Some regulators of osteogenesis, including growth factors and transcriptional factors, have been already identified, although further studies to better elucidate the molecular basis of this biological process are required in order to develop new and more effective therapeutic strategies for the treatment of osteogenic diseases, such as osteoporosis, bone fractures and osteosarcoma. Osteoblasts arise from mesenchymal stem cells (MSCs), which are adult multipotent stem cells that can be successfully isolated from several adult and neonatal tissues, are able to generate, following appropriate stimuli, several differentiated mesoderm-derived cell lineages, including adipocytes, osteoblasts, chondrocytes and myoblasts and, therefore, represent a promising cell source for regenerative medicine. In order to identify genes involved in the commitment of MSCs to osteoblasts, we performed a screening with an RNA interference-based approach and silenced a large number of genes during osteoblast differentiation of a murine MSC-derived cell line (W20-17). With this procedure we identified several candidate genes potentially involved in osteogenesis; in fact, their silencing caused a reduction of mineralized deposits, as assessed by Alizarin Red staining. We then analyzed the Gene Ontology classification of candidate genes and we found a significant fraction (30%) of genes with unknown function at the time of the screening. Hence, we focused our attention on one of these genes that we named Osteoblasts Inducer (ObI-1), predicted to be a transcription factor. Indeed, Ensembl analysis showed that ObI-1 encodes for a protein containing several zinc-finger domains and a Kruppel-associated box (KRAB) domain, usually involved in transcriptional repression. We were able to observe a significant impairment in osteoblast differentiation, resulting in reduction of both alkaline phosphatase expression and mineralization, as a result of ObI-1 silencing. In addition, we evaluated ObI-1 expression in our cell system as well as in several mouse tissues and organs. Interestingly, ObI1 expression increases during osteogenic differentiation of both W20-17 cell line and primary murine MSCs from bone marrow. We demonstrated also the nuclear localization of ObI-1 through immunofluorescence analysis, corroborating the hypothesis that this gene may encode for a transcription factor. Finally, we analyzed the effect of ObI-1 silencing on the expression of osteogenic markers during the differentiation process

Choroidal neovascularisation complicating geographic atrophy in age-related macular degeneration.

Abstract OBJECTIVE: To investigate the morphological and functional outcomes after intravitreal ranibizumab injections for choroidal neovascularisation (CNV) complicating geographic atrophy (GA). DESIGN: Retrospective, interventional, consecutive case series. METHODS: We reviewed the charts of all consecutive patients with GA due to age-related macular degeneration (AMD), who received intravitreal ranibizumab injections for the development of CNV at least 24 months earlier. RESULTS: 21 treatment-naive eyes of 21 consecutive patients (4 men, 17 women, mean age 86.9±1.6 years) were included. In 95.2% of eyes a type 2 CNV was present, extrafoveal in 42.8% of cases. After a mean of 5.0±0.87 (range 1-20) intravitreal ranibizumab injections, best-corrected visual acuity (BCVA) significantly worsened at the 24-month follow-up visit (0.73±0.05 vs 0.88±0.08 logMAR, respectively; p=0.01). A significant reduction of intraretinal cystic lesions, subretinal fluid and pigment epithelium detachment (p<0.001) and a significant increase of GA area (p=0.003) were present at last visit. CONCLUSIONS: Ranibizumab treatment of GA-associated CNVs provides no BCVA improvement at 24 months follow-up despite an anatomic response of CNV. Low effectiveness of ranibizumab in these cases is likely due to GA progression

Mechanisms and design of Tc1/mariner transposons for genome engineering

Transposons are DNA segments that autonomously move within and between genomes across the tree of life. Tc1/mariners in particular have frequently crossed species boundaries in nature and provide powerful broad-host-range genetic vectors. Among them, the Sleeping Beauty (SB) transposon inserts DNA in vertebrate genomes with extraordinarily high efficiency, making it a prime genetic tool with applications expanding to gene therapy clinical trials. Nevertheless, the molecular principles of SB’s distinctive activity remain elusive, greatly hampering its further development. In the first part of this thesis, I investigated the molecular mechanisms of the SB transposon in comparison to Human mariner 1 (Hsmar1), a representative transposon of the same superfamily. Using biochemical and biophysical techniques together with fluorescence-based assays, I have characterized the initial steps of SB and Hsmar1 transposition and shown that the two transposons assemble their molecular machineries (or transpososomes) differently. By combining crystallographic data and SAXS-based modelling, I visualized the structural basis of these differences and explained how transpososome assembly is coupled to catalysis in the Hsmar1 transposon. Moreover, the data demonstrated that the unique assembly pathway of SB largely contributes to its exceptional efficiency and that it can be chemically modulated to control insertion rates in living cells. I have further reconstituted in vitro the ordered series of events comprising SB transposition, including transposon end binding, cleavage, and integration, and dissected previously unrevealed molecular features of the process. In the second part of my work, building on these mechanistic insights, I developed a novel SB transposase variant (hsSB) by employing a structure-based protein design approach. Using hsSB allowed for establishing a new genome engineering method based on the direct delivery of recombinant SB protein to cells. We showed that this new method, named SBprotAct, provides safer and more controlled genome modification of several cell types (including stem cells and human T cells), as compared to the state-of-art technology. This work sheds first light on the molecular determinants of SB transposition and its hyper-activity, providing a unique resource for the rational design of improved genome engineering platforms for research and medicine

ophthalmology point of care a new journal for hands on clinicians

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The Evolution of the Plateau, an Optical Coherence Tomography Signature Seen in Geographic Atrophy

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Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells.

Osteosarcoma is a highly metastatic tumor affecting adolescents, for which there is no second-line chemotherapy. As suggested for most tumors, its capability to overgrow is probably driven by cancer stem cells (CSCs), and finding new targets to kill CSCs may be critical for improving patient survival. TP53 is the most frequently mutated tumor suppressor gene in cancers and mutant p53 protein (mutp53) can acquire gain of function (GOF) strongly contributing to malignancy. Studies thus far have not shown p53-GOF in osteosarcoma. Here, we investigated TP53 gene status/role in 3AB-OS cells-a highly aggressive CSC line previously selected from human osteosarcoma MG63 cells-to evaluate its involvement in promoting proliferation, invasiveness, resistance to apoptosis and stemness. By RT-PCR, methylation-specific PCR, fluorescent in situ hybridization, DNA sequence, western blot and immunofluorescence analyses, we have shown that-in comparison with parental MG63 cells where TP53 gene is hypermethylated, rearranged and in single copy-in 3AB-OS cells, TP53 is unmethylated, rearranged and in multiple copies, and mutp53 (p53-R248W/P72R) is post-translationally modified and with nuclear localization. p53-R248W/P72R-knockdown by short-interfering RNA reduced the growth and replication rate of 3AB-OS cells, markedly increasing cell cycle inhibitor levels and sensitized 3AB-OS cells to TRAIL-induced apoptosis by DR5 up-regulation; moreover, it strongly decreased the levels of stemness and invasiveness genes. We have also found that the ectopic expression of p53-R248W/P72R in MG63 cells promoted cancer stem-like features, as high proliferation rate, sphere formation, clonogenic growth, high migration and invasive ability; furthermore, it strongly increased the levels of stemness proteins. Overall, the findings suggest the involvement of p53-R248W/P72R at the origin of the aberrant characters of the 3AB-OS cells with the hypothesis that its GOF can be at the root of the dedifferentiation of MG63 cells into CSCs

Guidelines on Optical Coherence Tomography Angiography Imaging: 2020 Focused Update

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Prognostic relevance of optical coherence tomography angiography biomarkers in diabetic macular edema

Abstract was not mandatory. The review article was devoted to describe optical coherence tomography angiography (OCTA) biomarkers with a prognostic significance in predicting the evolution of diabetic macular edema (DME). OCTA features are crucial to the early prediction of DME but also to evaluate the response to treatment, identifying potential poor responders