Institute for Bioengineering and Biosciences

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Publications

Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

Publication . Arez, Maria; Eckersley-Maslin, Melanie; Klobuar, Tajda; von Gilsa Lopes, João; Krueger, Felix; Mupo, Annalisa; Raposo, Ana Cláudia; Oxley, David; Mancino, Samantha; Gendrel, Anne-Valerie; Jesus, Bruno Bernardes De; da Rocha, Simão T.

Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.

2022Journal article

Open access

In the flow of molecular miniaturized fungal diagnosis

Publication . Zolotareva, Maria; Cascalheira, Francisco; Caneiras, Catia; Bárbara, Cristina; Caetano, Diogo Miguel; Teixeira, Miguel Cacho

The diagnosis of fungal infections presents several challenges and limitations, stemming from the similarities in symptomatology, diversity of underlying pathogenic species, complexity of fungal biology, and scarcity of rapid, affordable, and point-of-care approaches. In this review, we assess technological advances enabling the conversion of cutting-edge laboratory molecular diagnostic methods to cost-effective microfluidic devices. The most promising strategies toward the design of DNA sequence-based fungal diagnostic systems, capable of capturing and deciphering the highly informative DNA of the pathogen and adapted for resource-limited settings, are discussed, bridging fungal biology, molecular genetics, microfluidics, and biosensors.

2024Journal article

Restricted access

Broad Spectrum Functional Activity of Structurally Related Monoanionic Au(III) Bis(Dithiolene) Complexes

Publication . Le Gal, Yann; Filatre-Furcate, Agathe; Lorcy, Dominique; Jeannin, Olivier; Roisnel, Thierry; Dorcet, Vincent; Fontinha, Diana; Francisco, Denise; Prudêncio, Miguel; Martins, Marta; Soeiro, Catarina; Sousa, Sílvia A.; Leitão, Jorge H.; Morais, Tânia; Bártolo, Inês; Taveira, Nuno; Guerreiro, Joana F.; Marques, Fernanda

The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/diselenolene) complexes were evaluated. The complexes differ in the nature of the heteroatom connected to the gold atom (AuS for dithiolene, AuSe for diselenolene), the substituent on the nitrogen atom of the thiazoline ring (Me, Et, Pr, iPr and Bu), the nature of the exocyclic atom or group of atoms (O, S, Se, C(CN)2) and the counter-ion (Ph4P+ or Et4N+). The anticancer and antimicrobial activities of all the complexes were investigated, while the anti-HIV activity was evaluated only for selected complexes. Most complexes showed relevant anticancer activities against Cisplatin-sensitive and Cisplatin-resistant ovarian cancer cells A2780 and OVCAR8, respectively. After 48 h of incubation, the IC50 values ranged from 0.1–8 µM (A2780) and 0.8–29 µM (OVCAR8). The complexes with the Ph4P+ ([P]) counter-ion are in general more active than their Et4N+ ([N]) analogues, presenting IC50 values in the same order of magnitude or even lower than Auranofin. Studies in the zebrafish embryo model further showed that, despite their marked anticancer effect, the complexes with [P] counter-ion exhibited low in vivo toxicity. In general, the exocyclic exchange of sulfur by oxygen or ylidenemalononitrile (C(CN)2) enhanced the compounds toxicity. Most complexes containing the [P] counter ion exhibited exceptional antiplasmodial activity against the Plasmodium berghei parasite liver stages, with submicromolar IC50 values ranging from 400–700 nM. In contrast, antibacterial/fungi activities were highest for most complexes with the [N] counter-ion. Auranofin and two selected complexes [P][AuSBu(=S)] and [P][AuSEt(=S)] did not present anti-HIV activity in TZM-bl cells. Mechanistic studies for selected complexes support the idea that thioredoxin reductase, but not DNA, is a possible target for some of these complexes. The complexes [P] [AuSBu(=S)], [P] [AuSEt(=S)], [P] [AuSEt(=Se)] and [P] [AuSeiPr(=S)] displayed a strong quenching of the fluorescence intensity of human serum albumin (HSA), which indicates a strong interaction with this protein. Overall, the results highlight the promising biological activities of these complexes, warranting their further evaluation as future drug candidates with clinical applicability.

2022-06-27Journal article

Open access

Generation and characterization of induced pluripotent stem cell line (IBBISTi004-A) from an Angelman syndrome patient carrying a class II deletion of the maternal chromosome 15q11.2-q13

Publication . Maranga, Carina; Pereira, Carolina; Raposo, Ana Cláudia; Vieira, Adriana A.; Duarte, Sofia; Bekman, Evguenia; Milagre, Inês; da Rocha, Simão T.

Angelman Syndrome is a rare neurodevelopmental disorder caused by several (epi)genetic alterations. The patients present strong neurological impairment due to the absence of a functional maternal UBE3A gene in neurons. Here, we generated and characterized a new induced pluripotent stem cell (iPSC) line from a female child with Angelman syndrome harbouring a class II deletion. iPSCs were reprogrammed from fibroblasts using Sendai viruses. The new iPSCs express pluripotency markers, are capable of trilineage in vitro differentiation and have the expected imprinting status of Angelman syndrome. These iPSCs are a valuable tool to elucidate the pathophysiological mechanisms associated with this disease.

2022Journal article

Open access