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- Targeting tumour-related alternative splice variant RAC1B with anti-sense oligonucleotidesPublication . Bessa, Cláudia; Gonçalves, Vânia; Jordan, PeterOver 90% of human protein coding genes are able to generate more than one transcript due to alternative splicing. In cancer, certain alternative splicing variants are frequently overexpressed and contribute to tumour progression and aggressiveness. This is well illustrated by RAC1B, a variant of the small GTPase RAC1 that is overexpressed in tumours from colon, breast, lung, pancreas and thyroid. RAC1B is generated by inclusion of alternative exon 3b and the resulting protein contains 19 additional amino acids, which alter regulation and signalling properties. In colorectal cancer, RAC1B is overexpressed in the subgroup of BRAF mutated tumours. Using HT29 cells as a model, RAC1B was shown to exist predominantly in the active GTP-bound conformation in cells and signal via NF-κB to sustain tumour cell survival. To exploit the suitability of this highly gene-specific event for oligonucleotide-based therapeutic intervention, we tested different anti-sense oligonucleotides to interfere with RAC1B expression levels in cancer cells. We found that synthetic siRNAs that induced transcript degradation were able to efficiently suppress RAC1B transcript and protein levels in cells. Phosphorodiamidate morpholino-modifed oligonucleotides (PMO, morpholinos) present higher affinity and stability than conventional oligonucleotides. Thus, a morpholino was designed that targets the intron3–exon3b splice junction including the exonic splice enhancer sequence for SRSF1 binding that we previously identified. When validated in HT29 colorectal tumour cells, this morpholino performed poorly on levels of RAC1B transcript or protein and did not affect RAC1B-dependent cell viability. Indeed, morpholinos do not easily enter mammalian cells in culture but have shown efficacy in animal models in vivo and in human clinical trials. In order to validate, whether the morpholino’s target sequence was able to mediate RAC1B inhibition, we employed the nuclease resistant 2′-O-methyl-modified (2OM) oligonucleotides directed to this and three other exon 3b sequences. We found that all four 2OM oligos reduced RAC1B transcript and protein, comparable to the efficiency of the synthetic siRNA and sustained the downregulating effect for up to 72 h post-transfection.
- Characterization of Haemophilus influenzae from healthy children attending day-care centers in the Lisbon región, 2015-2019Publication . Bajanca Lavado, Maria Paula; Cavaco, Luís Filipe; Fernandes, Mariana; Touret, Tiago; Candeias, CatarinaHaemophilus influenzae colonizes the human upper respiratory tract, where it can remain asymptomatically. It can also progress from colonizer to pathogen and cause mucosal or invasive infections. The aim of this study was to unravel epidemiological aspects of H. influenzae nasopharyngeal colonization in healthy children attending day-care centers in Portugal. Methods Between 2015 and 2019, 1518 nasopharyngeal samples were collected from children up to six years old, attending day-care centers in Lisbon region. Samples were cultured in chocolate agar with bacitracin and isovitalex and screened for the presence of H. influenzae based on colony morphology. Pure cultures were obtained and capsular serotype was determined by PCR. β-lactamase production was assessed with nitrocefin. Antibiotic susceptibility was determined for all β-lactamase producer isolates and all encapsulated isolates by a microdilution assay. Genetic characterization based on whole genome sequencing (WGS) analysis was performed for encapsulated isolates. Results H. influenzae was presumptively identified in 1280 samples indicating a high carriage rate (84.3%). Of these, most isolates (96.7%) were non-encapsulated (NT). The 42 encapsulated isolates belonged to serotypes a (n=4), b (n=1), e (n=14), and f (n=23). A total of 7.5% (n=96) of the isolates were β-lactamase producers although a higher percentage (11.9%) was observed in encapsulated isolates. Most isolates were susceptible to all antimicrobials tested. MLST revealed low genetic variability among encapsulated isolates: Hia-ST23, Hib-ST6, Hie-ST18 and ST122; and Hif-ST124, ST973 and ST2346. Analysis of presence/absence of 105 virulence genes showed that this is associated with the serotype, with serotype b isolates having the highest number of virulence genes. Virulence genes associated with specific structures or functions, such as iron acquisition, adherence, biofilm development or immune evasion, were present in all isolates. Conclusion Our results show that, in children attending day-care centers in the Lisbon region, the proportion of H. influenzae carriers is high and that circulation of encapsulated isolates is rare. Characterization of circulating isolates is important for community surveillance as these isolates may progress to cause severe invasive disease.