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- NMR-based urinary biomarkers in pediatric primary mitochondrial disorders and chronic kidney disease: shared mitochondrial dysfunction, diverging biosignaturesPublication . Coelho, Margarida Paiva; Rodrigues, João E.; Costa, Teresa; Dias, Aureliano; Graça, Inês C. R.; Rocha, Hugo; Vilarinho, Laura; Martins, Esmeralda; Gil, Ana M.; Carvalho de Azevedo Rocha, Hugo DanielRenal involvement is a recognized feature of primary mitochondrial disorders (PMD), either at presentation or during the disease course. Simultaneously, the metabolomic fingerprint of chronic kidney disease (CKD) is often associated with underlying mitochondrial dysfunction. This study aimed to characterize urinary metabolic signatures in genetically confirmed paediatric PMD without chronic kidney disease, comparing them to healthy controls, suspected (unconfirmed) mitochondrial disease (SMD), and non-mitochondrial CKD. We performed untargeted 1 H NMR metabolomic profiling of 76 urine samples from 51 paediatric patients and 10 healthy controls. PMD patients in acute decompensation or known CKD and statistical outlier samples were excluded. Final comparisons included genetically confirmed PMD without CKD (n=13), SMD (n=10), non-mitochondrial CKD (n=28; 17 at stages 1–2 and 9 at stages 3–5), and healthy controls (n=10). Spectral data were analyzed using multivariate statistical approaches—including principal component analysis (PCA) and partial least squares–discriminant analysis (PLS-DA)—as well as univariate methods with Mann-Whitney U for pairwise group metabolite comparison. Urinary metabolic profiles of PMD patients differed from healthy controls and CKD patients. Multivariate analysis revealed a strong discriminative ability between PMD and controls (Q² = 0.53) and advanced CKD (Q2=0.78). Compared to controls, PMD patients had increased levels of Krebs cycle intermediates (cis-aconitate, fumarate and succinate), creatine, tryptophan, homovanillate (HVA) and hypoxanthine, as well as decreased histidine. All, except fumarate and histidine, remained discriminative when comparing PMD to CKD. CKD patients showed a diverging metabolomic fingerprint with 1-methylnicotinamide (MNA) and 2-hydroxyisobutyrate emerging as potential CKD-specific biomarkers, effectively discriminating between CKD stage 3–5 from earlier stages and controls. A five-metabolite panel comprising cis-aconitate, fumarate, HVA, tryptophan and histidine achieved high diagnostic performance for identifying PMD, with an area under the curve (AUC) of 0.836 (PMD vs. controls) and AUC=0.783 across all groups. This biosignature integrates metabolites involved in distinct functional domains including energy metabolism, neurotransmitter turnover and amino acid metabolism and renal handling. Urinary metabolomic profiling by NMR revealed a distinct biosignature in pediatric PMD patients without renal involvement, characterized by elevated levels of tryptophan, HVA, and Krebs cycle intermediates, and diminished histidine. The divergent changes in tryptophan, histidine and HVA, suggest a mitochondria-specific metabolic phenotype in PMD. These findings support the use of urinary NMR metabolomics as a non-invasive tool for biomarker discovery in PMD and highlight the potential of integrated, multiparametric metabolic fingerprints for diagnostic refinement and patient stratification.
- Meningeal γδ T cells facilitate bacterial entry into the brain in neonatal meningitis and trigger long-term behavioural sequelaePublication . Lorga, Inês; Soares, Joana; Bravo, Joana; Mesquita, Pedro; Ribeiro, Nuno; Rodrigues, João; Hassan, Ahmed; Mesquita, Morgana; Cavadas, Bruno; Georgiev, Hristo; Magalhães, Ana; Summavielle, Teresa; Vilanova, Manuel; Ribot, Julie C.; Andrade, Elva Bonifácio; Bonifácio Andrade, Elva; Summavielle, TeresaNeonatal bacterial meningitis is a life-threatening condition and a leading cause of neurodevelopmental impairment among survivors. Despite its prevalence, the role of meningeal immunity on disease pathology during early life remains largely unexplored. Using a clinically relevant mouse model of neonatal group B streptococcal meningitis and single-cell RNA sequencing, we observed that IL-17A (IL-17)-producing γδ T cells (γδ17 T cells) accumulate in the meninges during the acute phase of infection and persist throughout the lifespan. Importantly, mice deficient in γδ T cells or in IL-17 show a significantly lower bacterial colonisation in the brain parenchyma, and IL-17 neutralisation in the cerebrospinal fluid leads to a similar phenotype. Reduced blood-brain barrier permeability in the absence of γδ T cells results in decreased bacterial invasion and diminished microglia activation. Wild-type but not γδ T cell-deficient mice surviving infection exhibit increased hyperactivity and open space anxiety during early adulthood, a behavioural profile that may reflect attention deficit and hyperactivity (ADHD)-like tendencies. Altogether, these findings establish a pathogenic role for meningeal γδ17 T cells in early life, uncovering a key mechanism that drives long-term sequelae in GBS neonatal meningitis.
- CGRP suppresses protective SiglecFhi Neutrophil development in Neonatal Group B Streptococcus PneumoniaPublication . Lorga, Inês; Teixeira, Ana Sofia; Carvalho, Bárbara; Soares, Joana; Ribeiro, Nuno; Cardoso, Marcos S.; Cunha, Joana; Santos, Joana; Silva, Regina A.; Vilanova, Manuel; Andrade, Elva Bonifácio; Bonifácio Andrade, Elva; Silva, ReginaNeonatal pneumonia, a leading cause of morbidity and mortality, is frequently caused by Group B Streptococcus (GBS). The mechanisms underlying protective immunity to this pathogen in the neonatal lung remain incompletely understood. Using a clinically relevant neonatal mouse model of GBS pneumonia, we investigated the immune mechanisms influencing disease severity. We demonstrate that neutrophils are effectively recruited to the lungs of infected neonates, but their phenotype differs with disease severity. In pups with moderate disease, we observe significant infiltration of SiglecFhi neutrophils, a phenotype associated with enhanced phagocytic capacity and bacterial clearance. In contrast, pups with severe disease failed to develop SiglecFhi neutrophils, resulting in reduced bacterial clearance and worsened pathology. We further show that severity is associated with increased expression of calcitonin gene-related peptide (CGRP) in the lungs. CGRP suppressed neutrophil activation into the SiglecFhi phenotype, thereby limiting their antibacterial function. Our findings show that GBS exploits the neuroimmune axis to evade host immunity through CGRP-mediated suppression of neutrophil activation.
- 3-Bromopyruvate boosts the effect of chemotherapy in acute myeloid leukemia by a pro-oxidant mechanismPublication . Vieira, Joana Pereira; Preto, Ana; Granja, Sara; Queirós, Odília; Celeiro, Sónia Pires; Ko, Young Hee; Casal, Margarida; Matos, Catarina Barbosa; Baltazar, Fátima; Granja, SaraAcute myeloid leukemia (AML) comprises a diverse group of blood cancers with varying genetic, phenotypic, and clinical traits, making development of targeted therapy challenging. Metabolic reprogramming in AML has been described as relevant for chemotherapy effectiveness. 3-Bromopyruvate (3-BP) is an anticancer agent that undermines energy metabolism of cancer cells. However, the effect of 3-BP in hematologic malignancies, such as AML, needs further investigation. Thus, we aimed to explore 3-BP as a chemo-sensitizing agent in AML. Different approaches of combining 3-BP with classical chemotherapy (daunorubicin and cytarabin) were tested in diverse AML cell lines. Cell sensitivity to the different drug combinations was analyzed by Trypan blue staining. The effect of pre-treatment with a non-toxic concentration of 3-BP was assessed on the AML cell metabolic profile (Western blot and immunofluorescence), mitochondrial activity (cytometry flow), and antioxidant capacity (colorimetric detection kit). KG-1 and MOLM13 cells showed increased sensitivity to chemotherapy (decreased EC 50 values) after exposure to a non-toxic concentration (5 μ M) of 3-BP. In both cell lines, 5 glucose consumption without changing extracellular lactate levels. 5 μ μ M 3-BP decreased M 3-BP treatment increased reactive oxygen species levels and decreased cell antioxidant capacity by depleting reduced glutathione levels in both KG-1 and MOLM13 cells. Our results demonstrate that non-toxic concentrations of 3-BP enhance the effect of classical chemotherapy in AML cells through a pro-oxidant mechanism. These data unveiled a new approach for AML treatment, using 3-BP or other pro-oxidant agents as co-adjuvants of chemotherapy, subsiding chemotherapy- induced side effects.
- MCT1 is a new prognostic biomarker and its therapeutic inhibition boosts response to Temozolomide in human glioblastomaPublication . Gonçalves, Vera Miranda; Gonçalves, Céline S.; Granja, Sara; Castro, Joana Vieira de; Reis, Rui M.; Costa, Bruno M.; Baltazar, Fátima; Granja, SaraGlioblastoma, the brain tumour with highest prevalence and lethality, exhibits a characteristic glycolytic phenotype with increased lactate production. Recently, we reported a MCT1 overexpression in GBMs tumours, being associated to tumour growth and aggressiveness. Thus, we aimed to disclose the role of MCT1 in GBM prognosis and in vivo therapy response. Importantly, MCT1 overexpression is associated with poor prognosis of GBM. Moreover, MCT1 inhibition retards GBM tumour growth and boosts response to temozolomide treatment.
- Metabolism-targeted therapy in NSCLC – A new theranostics inhalation approach using lactate functionalized and selenium-chrysin loaded nanoparticles (SeChry@PUREG4-LA24)Publication . Mendes, Cindy; Martins, Filipa; Granja, Sara; Gonçalves, Joana; Barros, Hélio; Casimiro, Teresa; Ricardo, Ana Aguiar; Silva, Fernanda; Abreu, Bruna; Cristovão, Miguel; André, Saudade; Pereira, Sofia A.; Baltazar, Fátima; Marques, Helena Cabral; Gaspar, Maria Manuela; Gonçalves, Luís G.; Bonifácio, Vasco D. B.; Serpa, Jacinta; Granja, SaraLung cancer is one of the most lethal cancers globally, primarily due to delayed diagnosis and lack of specific and effective therapy. Increased lactate production and consumption, along with cysteine metabolic reliance, are features identified in NSCLC in our recent studies. Cancer metabolic remodeling leads to excessive ROS production, triggering oxidative stress, promoting angiogenesis, causing cellular and tissue damage, and contributing to various pathophysiological changes. This study aimed to investigate the therapeutic potential of selenium–chrysin (SeChry), a cysteine metabolism inhibitor, and its delivery targeted at MCT1 by encapsulation in fourth-generation polyurea dendrimers functionalized with lactic acid (PUREG4-LA24), the nanoformulation SeChry@PUREG4-LA24, in NSCLC. We explored the impact of SeChry nanoformulation on cell death mechanisms, including ferroptosis, and its influence on angiogenesis in in vitro and in vivo models. SeChry@PUREG4-LA24 induces cell death through the induction of intracellular ROS and lipid peroxides, resulting in distinct expression patterns of ferroptosis-associated genes across cell lines. Experiments using chicken embryo chorioallantoic membrane (CAM) and mouse orthotopic xenograft models revealed a trend toward decreased tumor growth and angiogenesis with SeChry@PUREG4-LA24 administration. These findings suggest the potential of SeChry@PUREG4-LA24 as an innovative therapeutic approach for NSCLC, highlighting its impact on cell death mechanisms and anti-angiogenic effects.
- String/Cdc25 phosphatase is a suppressor of Tau-associated neurodegenerationPublication . Oliveira, Andreia C.; Santos, Madalena; Pinho, Mafalda; Lopes, Carla S.Tau pathology is defined by the intracellular accumulation of abnormally phosphorylated Tau (MAPT) and is prevalent in several neurodegenerative disorders. The identification of modulators of Tau abnormal phosphorylation and aggregation is key to understanding disease progression and developing targeted therapeutic approaches. In this study, we identified String (Stg)/Cdc25 phosphatase as a suppressor of abnormal Tau phosphorylation and associated toxicity. Using a Drosophila model of tauopathy, we showed that Tau dephosphorylation by Stg/Cdc25 correlates with reduced Tau oligomerization, brain vacuolization and locomotor deficits in flies. Moreover, using a disease mimetic model, we provided evidence that Stg/Cdc25 reduces Tau phosphorylation levels independently of Tau aggregation status and delays neurodegeneration progression in the fly. These findings uncover a role for Stg/Cdc25 phosphatases as regulators of Tau biology that extends beyond their well-characterized function as cell-cycle regulators during cell proliferation, and indicate Stg/Cdc25-based approaches as promising entry points to target abnormal Tau phosphorylation.
- Inking cell blocks improves scanner detection for diagnosis in pathologyPublication . Eloy, Catarina; Neves, Beatriz; Vale, João; Campelos, Sofia; Curado, Mónica; Polónia, AntónioCell blocks may be hard to be totally automatically detected by the scanner (ADS),generating incomplete whole slide images (WSIs), with areas that are not scanned,leading to possible false negative diagnosis. The aim of this study is to test if inkingthe cell blocks helps increasing ADS. Test 1: 15 cell blocks were sectioned, one halfinked black (1HB) and the other inked green (1HG). Each of the halves was individu-ally processed to generate a WSI stained by the H&E. 1HBs and 1HGs had similarscanning time (median 59 s vs. 65 s, p = .126) and file sizes (median 382 Mbvs. 381 Mb, p = .567). The black ink interfered less in the observation (2.2%vs. 44.4%; p < .001) than in the green one. Test 2: 15 cell blocks were sectioned, onehalf inked black (2HB) and the other left unstained/null (2HN). Each of the halveswas individually processed to generate three WSIs—one HE, one periodic-acid Schiff(PAS), and one immunostained by cytokeratin AE1&AE3 (CKAE1AE3). HE and PASWSIs from both 2HN and 2HB groups were all totally ADS and had similar scanningtimes and file sizes. Concerning immunostaining with CKAE1AE3: ADS (46.7%vs. 93.3%; p = .014), median time for scanning (57 s vs. 83 s; p < .001) and file size(178 Mb vs. 338 Mb; p < .001) were reduced significantly in the 2HN group in com-parison with the 2HB. Although increasing scanning time and file size, inking the cellblocks helps increasing ADS after immunostaining, improving the safety and effi-ciency of the digital pathology workflow.
- One nodule–one punction–one slide: Optimizing thyroid fine‐needle aspiration for a digital workflowPublication . Maia, Ariana; Carvalho, Bárbara; Vale, João; Curado, Mónica; Ryan, Carmel; Polónia, António; Eloy, CatarinaInterventional pathologists have expanded their expertise by acquiring proficiency in ultrasound-guided thyroid fine-needle aspiration biopsy (FNAB) and are now required to optimize punction procedures due to low resources and digital workflows. The aim of this study is to compare FNAB sample adequacy in two series with one versus two slides available for cytopathological analysis and its influence on diagnosis categorization, time taken to reach a final diagnosis, scanning time and size of the digital files produced. Patients were retrospectively selected based on the sampling of thyroid nodules using either two glass slides (two-slide group) or one slide only (one-slide group) and cytological diagnosis was performed using the second edition of the Bethesda system. For each group, the initial 15 cases were sorted to be scanned. From a total of 713 procedures, 328 were sampled into two slides and 385 on one slide only. No significant differences were found regarding nodule size, location or EU-TIRADS classification between the two groups. The one-slide group did not exhibit a higher prevalence of non-diagnostic or atypia of undetermined significance (AUS) categories. As expected, the mean time taken to finalize diagnoses in cases where only one slide was prepared was 1.2 days faster. Scanning time and total file size were also significantly smaller in the one-slide group. Adopting the ‘one nodule–one puncture–one slide’ strategy for thyroid FNAB optimization enhances procedural efficiency in digital workflows, leading to cost savings without compromising diagnostic accuracy.
- Optimizing the management of thyroid specimens to efficiently generate whole slide images for diagnosisPublication . Eloy, Catarina; Vale, João; Barros, Mariana; Oliveira, Diana; Mesquita, Morgana; Curado, Mónica; Pinto, João; Polónia, AntónioTransition from optical to digital observation requires an additional procedure in the pathology laboratory, the scanning of glass slides, leading to increased time and digital archive consumption. Thyroid surgical samples often carry the need to collect several tissue fragments that generate many slides to be scanned. This study evaluated the impact of using different inking colours for the surgical margin, section thickness, and glass slide type, in the consumption of time and archive. The series comprehended 40 nodules from 30 patients, including 34 benign nodules in follicular nodular disease, 1 NIFTP, and 5 papillary carcinomas. In 12 nodules, the dominant pattern was microfollicular/solid and in 28 it was macrofollicular. Scanning times/mm2 were longer in red-inked fragments in comparison to green (p=0.04) and black ones (p=0.024), and in blue-inked in comparison to green ones (p=0.043). File sizes/mm2 were larger in red-inked fragments in comparison to green (p=0.008) and black ones (p=0.002). The dominant pattern microfollicular/solid was associated with bigger file size/ mm2 in comparison with the macrofollicular one (p