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- Establishment of a suppression therapy for beta-thalassemia due to a nonsense mutationPublication . Dias, Patrícia; Romão, LuísaBeta-thalassemia is a genetic disorder caused by the absence or reduction of human beta-globin protein levels, which causes a reduction on hemoglobin synthesis. This reduction can be caused by a premature termination codon (PTC) in the human beta-globin gene. The messenger ribonucleic acid (mRNA) containing a PTC can be recognized and degraded by the nonsense-mediated decay mechanism (NMD), if the PTC is located 50-54 nucleotides upstream of the last exon-exon junction. If the PTC is located near to the initiation codon (generally an AUG) or downstream of the last exon-exon junction, then this mRNA is not degraded by NMD. In the first case a small peptide is degraded by proteolysis and in the second case a long truncated protein is synthesised. These proteins aggregate and precipitate, leading to a severe damage of erythroid precursors. The conventional therapies for beta-thalassemia temporarily restore the levels of human beta-globin protein, however, these therapies have secondary effects that lead to the deterioration of the patient health. Given this, a therapy that could restore the levels of human beta-globin protein by inducing the readthrough of the PTC in human beta-globin gene, would be an advantage to these patients. Suppression therapy has been studied in detail for genetic diseases caused by PTCs. Salvatori et al. demonstrated the capability of G418 to induce the readthrough of a PTC at codon 39 of human beta-globin gene, however, the capability of suppression by this aminoglycoside at other codons of human beta-globin gene, and the use of other suppression compounds, have not yet been investigated. Suppression therapy consists in the readthrough of a nonsense codon into a sense codon, which allows the mRNA to be totally translated, resulting in a complete protein with partial or complete function. There are some compounds, including aminoglycosides and non-aminoglycosides, that bind to the decoding center of the ribosome and allow the near-cognate aminoacyl-tRNA binding, resuming translation. The main aims of this project were to understand if G418, an aminoglycoside, or PTC124, a non-aminoglycoside, produce efficient levels of readthrough of PTCs in human beta-globin mRNA, and to understand how different PTCs on beta-globin mRNA respond to the suppression therapy. Therefore, HeLa and HEK293 cells were transiently transfected with constructs containing the first 55 codons of human beta-globin gene fused to firefly luciferase gene (betaWT-FLUC, beta15-FLUC or beta39-FLUC). The results were obtained by Western blot and bioluminescence assays. The results obtained by Western blot seem to indicate that G418 induce the readthrough of PTCs at codon 15 or 39 of human beta-globin gene in HEK293 treated with concentrations higher than 400 microg/mL. Additionally, a PTC at codon 15 of human beta-globin gene seems to respond more efficiently to the readthrough than a PTC at codon 39. The results obtained for PTC124 are not conclusive, however, PTC124 might be inducing the readthrough of a PTC at codons 15 and 39 of human beta-globin gene in HEK293 cells treated with 5 microM, or 5, 10 and 15 microM of PTC124, respectively. Since G418 revealed to be able to restore the full-length human beta-globin protein in constructs containing a PTC at codon 15 and 39 of human beta-globin gene, it arises as a promising compound to be used in a future therapy for beta-thalassemia. Before that, it is important to study the effect of NMD in suppression therapy and how its inhibition can enhance the suppressive effect.
- Reduction in undiagnosed HIV infection in the European Union/European Economic Area, 2012 to 2016Publication . van Sighem, Ard; Pharris, Anastasia; Quinten, Chantal; Noori, Teymur; Amato-Gauci, Andrew J; ECDC HIV/AIDS Surveillance And Dublin Declaration Monitoring NetworksIt is well-documented that early HIV diagnosis and linkage to care reduces morbidity and mortality as well as HIV transmission. We estimated the median time from HIV infection to diagnosis in the European Union/European Economic Area (EU/EEA) at 2.9 years in 2016, with regional variation. Despite evidence of a decline in the number of people living with undiagnosed HIV in the EU/EEA, many remain undiagnosed, including 33% with more advanced HIV infection (CD4 < 350 cells/mm3).