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- Day-Zero Serum FTIR Spectroscopy Identifies a Biochemical Signature Associated with Functional Pancreas Graft Dysfunction After Simultaneous Pancreas–Kidney TransplantationPublication . Vigia, Emanuel; Ramalhete, Luís; Araújo, Rúben; Corado, Sofia; Barros, Inês; Chumbinho, Beatriz; Nobre, Ana; Carrelha, Sofia; Pico, Paula; Rodrigues, Fernando; Bigotte, Miguel; Magriço, Rita; Cotovio, Patrícia; Caeiro, Fernando; Aires, Inês; Silva, Cecília; Pena, Ana; Bicho, Luís; Jorge, Cristina; Calado, Cecília R.C.; Pereira, Jorge P.; Ferreira, Aníbal; Marques, Hugo P.; iNOVA4Health - pólo NMS; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); MDPI - Multidisciplinary Digital Publishing InstituteBackground: Simultaneous pancreas–kidney (SPK) transplantation can restore renal function and insulin independence, but non-technical pancreas graft dysfunction remains difficult to anticipate. Methods: We conducted an exploratory single-centre retrospective biomarker-modelling study to determine whether day-zero recipient serum Fourier-transform infrared (FTIR) spectra are associated with subsequent loss of insulin independence after SPK transplantation. Results: Among 104 screened recipients, 51 met predefined sample-availability, spectral-quality, data-linkage and endpoint-adjudication criteria; 30 maintained pancreas graft function and 21 developed dysfunction. Cases dominated by early technical surgical failure were excluded. Clinical-only, FTIR-only and FTIR–clinical Naïve Bayes models were evaluated using leave-one-out cross-validation with Fast Correlation-Based Filter feature selection. In locked-feature internal validation, the best FTIR-only model used second-derivative spectra with vector normalization and nine selected wavenumbers, achieving AUC 0.997 (95% CI 0.985–1.000) and accuracy 0.961 (95% CI 0.902–1.000). A fixed-feature permutation analysis exceeded label-randomized performance (empirical p = 0.001). The secondary Group 1 versus Group 3 analysis suggested discrimination of pancreas dysfunction despite preserved kidney function (AUC 0.992; accuracy 0.930). Conclusions: Given the small cohort, high-dimensional input, non-nested feature selection, selection-bias risk and absence of external validation, serum FTIR should be considered a candidate risk-enrichment platform requiring prospective multicentre validation.
- Enhancing lysosome function via TFEB activation reduces lipofuscin-like granules in the retinal pigment epitheliumPublication . Falcão, Ana S.; Lopes-da-Silva, Mafalda; Antas, Pedro; Escrevente, Cristina; Pedro, Margarida; Felgueiras, Beatriz; Caires, Beatriz; Coelho, Rita; Ferreira, Inês S.; Santos, Inês P.; Ciossek, Thomas; Nicklin, Paul; Tenreiro, Sandra; Seabra, Miguel C.; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); iNOVA4Health - pólo NMS; ElsevierAge-related macular degeneration (AMD) is the most common blinding disease in the western world and is currently incurable. Although the exact causes of AMD are not clear, the primary origin of pathology appears to be the aged retinal pigment epithelium (RPE) exhibiting signs of lysosomal dysfunction and oxidative damage. RPE is responsible for the daily digestion of photoreceptor outer segments (POS), imposing a heavy continuous burden on the lysosomal network. A cellular model of RPE lysosomal dysfunction can be achieved by feeding RPE with a single pulse of POS, leading to the accumulation of autofluorescence granules (AFG), similar to lipofuscin in vivo. Here we show that synchronous phagocytosis of POS leads to early transient mTOR activation followed by inhibition in late phagosome maturation. One of its substrates, the transcription factor EB (TFEB) increases during phagosome maturation albeit mostly in its inactive phosphorylated form. We questioned whether modulation of the mTOR/TFEB axis could improve POS clearance and hence reduce AFG load. Treatment of POS-fed cells after the appearance of AFGs with rapamycin, an mTORC1 inhibitor results in ∼30% reduction of AFG load. This effect is dependent on active lysosomal enzymes and induction of active dephosphorylated TFEB with consequent activation of GADD34 and lysosomal biogenesis. As a proof of concept, we show that overexpressing a constitutively active form of unphosphorylated TFEB dramatically reduces POS-dependent AFG accumulation. Overall, this study suggests that viral or pharmacological approaches activating the TFEB pathway in the RPE could be beneficial as cell-protective treatment of early/intermediate cases of AMD, acting to delay progression of the disease.
- The transformative impact of stem cell core facilities in biomedical researchPublication . Corneo, Barbara; Faga, Giovanni; Figueira, Inês; Zasso, Jacopo; Yang, Wenli; Neuman, Katrin; Johansson, Pia A.; Healy, Lyn; Daheron, Laurence M.; Generali, Melanie; Pless, Ole; Trokovic, Ras; David, Laurent; Gazhvini, Mehrnaz; Batlle-Morera, Laura; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); iNOVA4Health - pólo NMS; NOVA Institute for Medical Systems Biology; ElsevierOver the past three decades, advances in human pluripotent stem cell (hPSC) technologies, including induced pluripotent stem cells, gene editing, and 2D/3D models, have transformed biomedical research. These powerful tools have revolutionized disease modeling, drug discovery, and the development of advanced therapy medicinal products (ATMPs), while driving the establishment of stem cell core facilities. By providing specialized expertise, standardized workflows, and access to advanced technologies, these facilities support both fundamental and translational research, promote rigor and reproducibility, and foster collaboration. This manuscript highlights their role as hubs of excellence and discusses current challenges and future opportunities for the global stem cell community.
- Folic acid prevention of neural tube defects requires retinoic acid produced by ALDH1L1Publication . Edri, Tamir; Abbou-Levy, Tali; Cohen, Dor; Inácio, José M.; Shabtai, Yehuda; Pillemer, Graciela; Belo, José António; Fainsod, Abraham; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); iNOVA4Health - pólo NMS; National Academy of SciencesFolic acid (FA) supplementation during pregnancy is the commonly accepted treatment to prevent neural tube defects. The mechanism by which FA prevents neural tube defects (NTDs) remains unclear. FA also prevents other developmental malformations, including alcohol-induced malformations in Fetal Alcohol Syndrome models. We show that FA acts through a metabolic link to retinoic acid (RA) signaling. Using a pax3-knockdown Xenopus model of FA-rescuable NTDs, we show that RA or its precursors equally rescue these defects. Similarly, FA rescues alcohol-induced NTDs in a model previously shown to be rescued by retinoids. We identify the FA-metabolizing enzyme, formyl tetrahydrofolate dehydrogenase (ALDH1L1, FTHFD), encoded by the aldh1l1 gene, as essential for this rescue. Mechanistically, FA upregulates aldh1l1 expression, thereby increasing RA biosynthesis. Knockdown of ALDH1L1 activity using CRISPR/Cas9 abolishes the FA protective effect. To support these observations, we show that the human ALDH1L1 enzyme converts retinaldehyde to RA, and its overexpression restores neural tube closure in aldh1l1-knockdown embryos when retinaldehyde is provided. At the cellular level, reduced RA signaling results in overproliferation of neural plate precursors and a pathological expansion of the neural tube. ALDH1L1 enables FA to restore normal neural plate proliferation, thereby preventing NTDs. These findings establish ALDH1L1 as an unexpected enzymatic link between FA (vitamin B9) and RA signaling, revealing how FA supplementation safeguards neural development and suggesting opportunities to refine strategies for NTD prevention.
- PON1 haplotypes show genotype-dependent associations with dysglycemia and metabolic liver risk beyond paraoxonase activityPublication . Batista-Herrera, Laura; Meneses, Maria João; Ribeiro, Rogério T.; Gardete-Correia, Luís; Raposo, João F.; Boavida, José Manuel; Penha-Gonçalves, Carlos; Macedo, Maria Paula; iNOVA4Health - pólo NMS; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Vector borne diseases and pathogens (VBD); Global Health and Tropical Medicine (GHTM); Laboratório Associado de Translacção e Inovação para a Saúde Global - LA Real (Pólo IHMT); Instituto de Higiene e Medicina Tropical (IHMT); Frontiers MediaIntroduction – Paraoxonase 1 (PON1) is a liver-derived HDL-associated enzyme with key antioxidant functions implicated in cardiometabolic disease. Genetic variation in PON1 strongly influences enzyme activity; however, whether specific genetic configurations contribute to dysglycemia and metabolic liver risk beyond enzymatic activity remains unclear. Methods – We analyzed 922 individuals from the PREVADIAB2 cohort to investigate the relationship between PON1 genetic variation, serum paraoxonase (PONase) activity, and dysmetabolic phenotypes. Genetic determinants of PONase activity were identified using genome-wide analysis. Independent variants were combined into haplotypes, and their associations with dysglycemia and metabolic liver risk (Fibrotic NASH Index, FNI) were assessed in individuals aged >55 years. Results – Two independent PON1 variants—rs2057681 (in strong linkage disequilibrium with Q192R) and the promoter variant rs854572—were identified as major determinants of PONase activity. Haplotype analysis revealed that promoter–transcribed region combinations exert graded effects on enzyme activity. Importantly, these genetic configurations were differentially associated with dysglycemia and metabolic liver risk in a genotype-dependent manner. In carriers of the rs2057681 G allele, the C–A haplotype was associated with lower risk of dysglycemia and elevated FNI, whereas in rs2057681 AA homozygotes the same haplotype showed an opposite association with metabolic liver risk. Notably, despite strong genetic effects on PONase activity, enzyme activity itself was not directly associated with dysmetabolic phenotypes. Discussion – PON1 genetic architecture, defined by promoter– transcribed region interactions, is associated with dysglycemia and metabolic liver risk in a genotype-dependent manner beyond steady-state enzyme activity. These findings provide insight into the genetic regulation of metabolic risk and may help explain inconsistent associations of PON1 variants in cardiometabolic disease.
- Risk Phenotyping Before Graft ImplantationPublication . Ramalhete, Luis; Araújo, Rúben; Vigia, Emanuel; Vieira, Miguel Bigotte; Ferreira, Anibal; Calado, Cecilia R.C.; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); iNOVA4Health - pólo NMS; MDPI - Multidisciplinary Digital Publishing InstituteBackground: Rejection remains a major barrier to long-term kidney allograft survival, and pre-transplant risk stratification remains incomplete. This study evaluated whether pre-transplant serum Fourier-transform infrared (FTIR) spectra, analyzed using machine learning methods, could identify kidney transplant recipients at increased risk of subsequent biopsy-proven rejection. Methods: In this retrospective single-center study, 80 pre-transplant serum samples collected on the day of transplantation were initially evaluated; after spectral quality control, 79 samples were retained for analysis. FTIR spectra were acquired in transmission mode and analyzed in the 600–1900 cm−1 and 2800–3400 cm−1 regions. Multiple preprocessing strategies were assessed, including Rubber Band baseline correction, vector normalization, and first- and second-derivative transformation, with and without normalization. Naïve Bayes classifiers with Leave-One-Out Cross-Validation and Fast Correlation-Based Filter feature selection were applied. Results: Exploratory analysis showed broad overlap between groups, indicating a subtle multivariate spectral signal. In the initial exploratory workflow, classifier performance depended strongly on preprocessing and feature selection. Because non-nested feature selection may produce optimistic estimates, the main supervised analysis was repeated using FCBF nested within each LOOCV training fold. The best-performing nested model was obtained using second derivative transformation followed by normalization in the combined 600–1900 and 2800–3400 cm−1 regions, achieving an AUC of 0.837, accuracy of 0.747, sensitivity of 0.675, specificity of 0.821, balanced accuracy of 0.748, and F1-score of 0.730. Permutation testing with 1000 label-randomized repetitions supported performance above chance expectation, with no permuted model reaching the observed AUC (empirical p = 0.000999). Conclusions: Pre-transplant serum FTIR spectroscopy combined with leakage-aware nested machine learning analysis identified an internally validated spectral signal associated with subsequent biopsy-proven rejection. These findings support FTIR as a promising complementary and hypothesis-generating approach for pre-transplant biochemical risk phenotyping, requiring external multicenter validation before clinical application.
- In silico prediction of the permeability of phytochemicals across the blood-brain barrierPublication . Carregosa, Diogo; Moreira, Iris; Rasteiro, Diogo; Vanneschi, Leonardo; Nunes dos Santos, Cláudia; Programme in Translational Medicine (iNOVA4Health); NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); iNOVA4Health - pólo NMS; NOVA Institute for Medical Systems Biology; NOVA Information Management School (NOVA IMS); Information Management Research Center (MagIC) - NOVA Information Management School; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Frontiers MediaIntroduction: The role of phytochemicals like xanthines, and (poly)phenols in neurodegenerative diseases has been highly established and explored. However, the mechanism of these molecules to cross the membranes reaching the blood and the brain has largely been understudied. Methods: In this work, we used in silico methods to predict the permeability of more than 800 molecules across the membranes and specifically the blood-brain barrier. Our dataset included phytochemicals, like xanthine, phenolic terpenes, (poly)phenols and metabolites, including phase II conjugates, while some endogenous molecules and commercial drugs were used as positive/negative controls. We used QikProp to generate 42 computed physicochemical properties to predict the blood absorption, distribution to the brain, metabolism, and elimination of these molecules. Results: According to Qikprop 555 of 800 molecules were inside the range of 95% of known drugs for all computed properties, while through passive di[usion, 78 molecules may reach the blood and 52 may reach the brain parenchyma. Furthermore, using data from natural molecules present in human CSF samples, we used machine learning to build a model that was capable to predict 171 novel molecules with the potential to reach the brain environment. Conclusion: Overall, in this work we predicted the natural molecules with the potential to reach the brain, highlighting those that may do so by passive mechanisms. Our study creates an opportunity to track novel molecules in theblood and the brain and highlighting the most drug-promising natural molecules for brain drug development in the treatment and prevention of brain diseases.
- mRNA delivery to the retina restores REP1 function in choroideremiaPublication . de Lemos, Luisa; Antas, Pedro; Carvalho, Cláudia; Castro, Mariana; Bruno, Diogo; Fonseca, Ana F; Rakshit, Shuvajit; Seabra, Miguel C; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); iNOVA4Health - pólo NMSInherited retinal diseases (IRDs) represent a major cause of blindness, yet current viral vector-based gene therapies are limited by high cost, restricted cargo capacity, and safety concerns related to immunogenicity. We explore here retinal delivery of non-viral, in-vitro-transcribed mRNA using lipid nanoparticles (LNPs) as a safe, cost-effective gene augmentation strategy for choroideremia (CHM). CHM is an X-linked IRD caused by loss-of-function mutations in CHM that encodes Rab escort protein-1 (REP1), which is essential for Rab GTPases prenylation and intracellular trafficking. We demonstrate that subretinal delivery of human CHM-mRNA (hCHM-mRNA) efficiently targets the retinal pigment epithelium (RPE) and choroid, achieving expression that is detectable up to 15 days post-injection, with lower and transient inflammatory response compared to AAV2 vectors. Furthermore, hCHM-mRNA delivery in vitro to human induced pluripotent stem cell (hiPSC)-derived RPE cells from a CHM patient and in vivo via subretinal injection in the Chm ± mouse model successfully restored REP1 levels and reversed Rab prenylation defect. Concordantly, Chm ± treated with hCHM-mRNA showed functional rescue of retinal activity measured by electroretinography 24 h post-injection. Overall, these results provide a strong proof-of-concept for LNP-mediated mRNA technology in the retina, supporting the development of scalable, non-viral gene approaches for IRDs, including CHM.
- Increased Expression of HLA-DR and CD69 on Peripheral CD4+ T Cells Predicts Better Clinical Outcomes in Cutaneous MelanomaPublication . Tomás, Ana; Maximino, José; Nunes, Hugo; Salvador, Rute; Luís, Rafael; Brito, Cheila; Saraiva, Diana P.; Gouveia, Emanuel; Pereira, Carolina; Gonçalves, Filipe; Farricha, Victor; Lopez Carvalho, Elisabete; Moura, Cecília; Passos, Maria José; Cristóvão-Ferreira, Sofia; Pereira, Patrícia M.; Cabral, Maria Guadalupe; Pojo, Marta; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); iNOVA4Health - pólo NMS; Dove Medical PressBackground: Cutaneous melanoma (CM) is an aggressive skin cancer with rising incidence, representing a growing public health concern. Despite the remarkable success of immune-checkpoint inhibitors (ICIs) in the management of advanced disease, mortality remains high due to therapy resistance. Identifying reliable prognostic and predictive biomarkers is therefore essential to improve patient stratification, optimize treatment selection, and minimize unnecessary toxicity. Methods: We comprehensively profiled the circulating immune landscape of 54 treatment-naïve CM patients by integrating flow cytometry immunophenotyping with clinicopathological data, and performed tumor gene expression analysis in a subset of 26 patients. Results: Elevated HLA-DR and CD69 expression on circulating CD4+ T cells, together with reduced circulating CD8+ T cell frequency, emerged as candidate prognostic biomarkers associated with improved survival. Prognostic models combining these immune variables with clinical covariates accurately stratified patients by overall survival (89.5% sensitivity, 72.7% specificity; AUC = 0.872, p < 0.0001) and progression/recurrence risk (75% sensitivity and 71.4% specificity; AUC = 0.763, p = 0.001). In a subset of 43 patients subsequently treated with ICIs, elevated baseline HLA-DR and CD69 expression on circulating CD4+ T cells was also associated with therapeutic benefit. A predictive model integrating these markers with clinical covariates achieved good discriminatory performance (65.2% sensitivity, 88.9% specificity; AUC = 0.775, p = 0.0027). Tumor gene expression profiling supported the role of IFN-γ-related signatures, previously linked to ICI response, as complementary prognostic and predictive tools. Conclusion: These findings highlight systemic CD4+ T cell activation status as a potential, easily measurable biomarker in CM, laying the foundation for future strategies to refine patient stratification and guiding immunotherapy decisions.
- Translational insights into extracellular vesicles in peritoneal dialysisPublication . Sequeira, Diogo; Anão, Sofia; Calça, Ana Rita; Matias, Patrícia João; de Melo Junior, Antonio Ferreira; Ferreira, João Vasco; Carvalho, Ana Sofia; Matthiesen, Rune; Branco, Patrícia; de Azeredo Pereira, Sofia; Sousa, Cátia; Teixeira-Santos, Luísa; iNOVA4Health - pólo NMS; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Multimed Inc.Peritoneal dialysis (PD) is a cornerstone kidney replacement therapy for patients with end-stage kidney disease; however, chronic exposure to bioincompatible dialysis solutions progressively damages the peritoneal membrane, leading to mesothelial-to-mesenchymal transition, fibrosis, and ultimately ultrafiltration failure. Currently, peritoneal membrane dysfunction is detected only at advanced stages through the peritoneal equilibration test, underscoring the need for earlier and more sensitive biomarkers. Extracellular vesicles (EVs) isolated from peritoneal dialysis effluent (PDE) have emerged as promising candidates, given their capacity to carry proteins, lipids, and nucleic acids that reflect local and systemic cellular activity. To date, 12 studies have successfully isolated and characterized PDE-derived EVs, identifying canonical EV markers, such as CD9, CD63, CD81, TSG101, and HSP70, alongside disease-relevant molecules, including galectin-3 binding protein, aquaporin-1, glycoprotein 96, and integrin-linked kinase. These EVs are enriched in signaling components associated with inflammation, angiogenesis, and fibrosis, particularly through TGF-β/p38 and NF-κB pathways. Moreover, specific EV-associated microRNAs (e.g., miR-125a-5p, miR-132-3p, miR-296-3p, miR-432-5p) overlap with molecular signatures observed in kidney and cardiometabolic disorders, suggesting broader systemic relevance. As an original contribution to this review, and to address cross-study comparability, we applied EVqualityMS, a mass spectrometry-based quality assessment tool, to benchmark EV enrichment and contaminant profiles and calculate quality indices across publicly available PDE-EV proteomics datasets. Despite methodological heterogeneity and limited sample sizes, PDE-derived EVs represent a powerful “liquid biopsy” of the peritoneal environment. Their integration into PD monitoring holds promise for the early detection of membrane injury and for supporting a predictive, biomarker-guided, and personalized approach to PD management.