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- Optimization of SPE-HPLC-FLD methodologies for the extraction and detection of steroid hormones in plasmaPublication . Bracchi, Isabella; Paíga, Paula; Delerue-Matos, Cristina; Pestana, Diogo; Negrão, Rita; Keating, Elisa; Fernandes, VirgíniaThe quantification of steroid hormones, such as es-tradiol and ethinylestradiol, in plasma requires sensi-tive and reliable analytical methodologies1. This study presents the optimization of a bioanalytical method based on Solid-Phase Extraction (SPE) coupled with High-Performance Liquid Chromatography with Flu-orescence Detection (HPLC-FLD). For sample ex-traction, Strata-X (polymeric reversed-phase) cartridg-es were used, where critical SPE parameters, including cartridge conditioning, matrix washing, and analyte elution, were evaluated. Chromatographic separation was achieved on a C18 stationary phase using a mobile phase of acetonitrile and 0.1% formic acid in ultrapure water. Both isocratic and gradient elution programs were tested to maximize resolution and peak shape. The method was validated for linearity, recovery, and precision (intra- and inter-day), meeting all established criteria for bioanalytical applications. This combined optimization resulted in improved selectivity and sen-sitivity for the determination of estradiol and ethinyl-estradiol in plasma (Figure 1), demonstrating a robust bioanalytical tool for monitoring these compounds in complex biological matrices.
- Evaluation of adherence to different pureed diets in a hospital contextPublication . Serdoura, Sara; Lopes, Mariana; Gomes, Isabel; Rodrigues, João Costa; Vieira, Mónica Almeida; Almeida Vieira, Mónica Andreia; Costa-Rodrigues, JoaoFood waste in hospital settings is higher than in other food sectors. Hospital diets constitute the main food and nutritional source for patients, so that incomplete food intake can have negative clinical consequences. In the pureed texture diet, foods are presented in a homogeneous, moist, cohesive manner and without lumps or pieces, resembling the texture of a pudding. The aim of present study was to evaluate adherence to a homogeneous and tri-compartmentalized pureed diet in adult patients admitted to a public hospital. Observational study to assess adherence to a creamy diet at lunch and dinner, on three different days. The pureed tri-compartmentalized diet dish was provided at lunch and a homogeneous diet at dinner. The tricompartmental diet plate presented individualized macronutrients (protein, carbohydrates and vegetables) and in the homogeneous diet these components were mixed. As regards the soup and dessert provided, there was no distinction between the meals. To quantify food waste, the physical method of individual weighing was considered. A satisfaction questionnaire was applied to determine patient preferences. A total of 152 complete meals were evaluated from 76 patients with a mean age of 78.9 years (SD=9.4), of which 51.3% were male (n=39). Plate waste in the homogeneous diet was 71.0% and in the tri-compartmentalized diet it was 65.8%. Regarding the waste of soup and dessert, it was greater at lunch than at dinner. It was found that 33.3% of patients preferred the tri-compartmentalized pureed diet due to its improved presentation and flavor, however 45.1% of patients reported having no preference regarding the presentation of the dish in this diet (Table 1). The high food waste of the pureed diet requires intervention, so presenting the meal plate in a tri-compartmentalized format appears to be a possible action to improve food intake.
- Food and lifestyle determinants of maternal exposure to semi-metal and heavy metal pollutantsPublication . Guimarães, Juliana; Bracchi, Isabella; Pinheiro, Cátia; Vieira, Maria Inês; Barbosa, Margarida Oliveira; Almeida, Agostinho; Azevedo, Rui; Pinto, Edgar; Pestana, Diogo; Calhau, Conceição; Prucha, Maria do Carmo; Martins, Cristina; Dias, Cláudia C.; Azevedo, Luís Filipe R.; Ramalho, Carla; Fernandes, Virgínia Cruz; Keating, Elisa; Fernandes, Virgínia; Pinto, EdgarExposure to toxics elements such as Lead (Pb), Cadmium (Cd), Lithium (Li) and Arsenic (As) pose health risks particularly for vulnerable groups like pregnant women and children. Knowledge about toxic metal exposure in these groups is scarce in Portugal. This study aims to identify food and lifestyle determinants of maternal exposure to toxic elements. Pregnant women from the IoMum cohort, undergoing firsttrimester fetal ultrasound between April 2018 and December 2021 at CHUSJoão (Porto) and Hospital CUF Descobertas (Lisbon), were invited to participate. At this stage, dietary and lifestyle information was collected through a structured questionnaire. A random urine sample was collected for Pb, Cd, Li and total (inorganic + organic) As quantification by ICP-MS and the values were adjusted for urinary creatinine concentration. A total of 305 mother: child pairs were included. RESULTS: The majority of the sample had detectable urinary levels of toxic elements (n (%): 269 (88), 157 (52), 305 (100) and 293 (96) for Pb, Cd, Li and total As, respectively). Pb urinary concentration was inversely associated with education level (p=0.008) while Cd and As levels were positively associated with education level (p=<0.001 and 0.002, respectively). Smoking was associated with higher Pb levels (p<0.001). Fish intake frequency was positively associated with Cd and As levels (p=0.005 and p<0.001, respectively) with women with fish intake higher than 4 times a week presenting median (P25;P75) in μg/g total As 33.8 (20.9 ; 67.7) concentration above the reference range typically (≥ 24.0 μg/g), seen in a group of healthy individuals. On the other hand, milk intake frequency was inversely associated Pb levels (p=0.025). Our results suggest that fish consumption may be a significant source of Cd and As exposure and that a higher milk intake may have a protective effect against Pb exposure.
- A nutritional intervention to promote mediterranean diet in high-risk pregnancies: Preliminary results from a randomized trialPublication . Gonçalves, Carlota; Morais, Juliana; Ferreira, Ana Filipa; Ramalho, Carla; Maia, Benedita Sampaio; Pires, Inês FalcãoThe rising prevalence of obesity during women’s reproductive years highlights the need for effective strategies to improve nutritional literacy as a preventive approach to adverse postpartum outcomes. This pilot randomized controlled trial aims to evaluate the impact of nutritional education sessions promoting the Mediterranean Diet (MedDiet) on Mediterranean Diet Adherence Screener (MEDAS), preconception weight recovery, and cardiometabolic outcomes. A PERIMYR-OralBioBorn mother-infant birth cohort sub-sample with obesity, hypertension, and/or diabetes was randomized to receive a MedDiet intervention, that consisted of a face-to-face session using a 13-page manual, followed by five monthly newsletters. Participants were re-evaluated at one, and six months postpartum. To assess the adherence to the MedDiet, the 14-item questionnaire MEDAS validated for the Portuguese population was used. Cardiac function and structure during pregnancy and postpartum were evaluated by echocardiography. Statistical analyses included Chi-square tests, T-tests, and Mann-Whitney tests. RESULTS: Fifty-six women with cardiometabolic risk factors (obesity, diabetes, and/or hypertension), with a median age of 33 [30; 36.5] years in the third trimester of pregnancy, were included. No significant differences in MedDiet adherence scores were observed between control and intervention groups before the intervention (8.6±1.8 vs. 8.5±1.5, p=0.815). One month postpartum, both groups maintained moderate adherence (7.7±2.1 vs. 8.2±2.6, p=0.731), with no differences between groups at six months (7.4±1.9 vs. 8.1±2.3, p=0.466). The intervention did not affect preconception weight recovery at six months. However, the intervention group showed better cardiac relaxation (E/e’ ratio) than controls (5.7±1.4 vs. 6.6±1.4, p=0.042), with no differences in cardiac mass or left ventricle wall thickness regression 6 months postpartum. Although preliminary findings do not indicate a significant change in MedDiet adherence scores or weight recovery, we observed improved left ventricular diastolic function. Future research incorporating detailed dietary intake assessments, metabolic biomarkers, and physical activity tracking could help clarify these effects.
- Impact of maternal stevia consumption on offspring`s metabolic health: A role in fetal programmingPublication . Bracchi, Isabella; Barbosa, Margarida; Vieira, Maria Inês; Brandão, Sofia; Esteves, Ana Carolina; Martins, Carina; Rocha, Inês; Pestana, Diogo; Negrão, Rita; Keating, ElisaThe consumption of non-sugar sweeteners (NSS) by pregnant women has increased in recent decades. Stevia, a natural NSS, is generally considered safe, but the World Health Organization recently recommended restricting the use of NSS, including stevia, highlighting the need to assess their safety during early stages of development. To investigate the role of RebaudiosideA (RebA) consumption, the main sweetener component of Stevia, as a fetal programmer of metabolic dysfunction. Female Sprague Dawley rats were administered RebA in the drinking water (4mg steviolequivalents/kg bodyweight/day, corresponding to the EFSA’s ADI) from 4 weeks before mating until weaning (total 13 weeks) (RebA, n=8), or water as control (C, n=8). Offspring (G1, both sexes) were weaned at 3 weeks of age into a standard diet until 10 months of age. G1 morphometry, food and water consumption were assessed. G1 glucose homeostasis was assessed by oral glucose tolerance (OGTT) and insulin sensitivity tests (IST) at 2, 6 and 10 months of age. Two-wayANOVA, with Sidak correction, was performed to test the effect of time, RebA, and the interaction between them. Female offspring early exposed to RebA (RebA-G1) presented greater weight gain from 30 weeks of age and onwards when compared to matched controls (p=0.0165). Food and water consumption were similar between groups. Early exposure to RebA changed the glycemic response of female G1 over time during the IST (p=0.0200) at 10 but not at 2 or 6 months of age. Male RebA-G1 presented higher glycemia 2h-post OGTT at 10 months of age, when compared to matched controls (p=0.0197). Maternal RebA consumption during the perigestional period led to increased weight gain and signs of insulin resistance in female offspring, and a degree of dysglycemia in male offspring. These findings strongly suggest a role of RebA in fetal programming of metabolic dysfunction.
- Determinants of maternal exposure to heavy metals and semi-metals: Focus on diet and lifestylePublication . Guimarães, J.; Bracchi, Isabella; Pinheiro, C.; Vieira, M.I.; Barbosa, M. Oliveira; Almeida, A.; Azevedo, R.; Pinto, Edgar; Pestana, D.; Calhau, C.; Prucha, M. do Carmo; Martins, C.; Dias, C.C.; Azevedo, L.F.R.; Ramalho, C.; Fernandes, Virginia Cruz; Keating, E.; Fernandes, Virgínia; Pinto, EdgarKnowledge on determinants and health risks of toxic metal exposure in pregnancy is scarce in Portugal. This study aims to characterize food and lifestyle determinants of maternal exposure to Lead (Pb), Cadmium (Cd) and Arsenic (As). We included 1111 pregnant women undergoing routine 1sttrimester fetal ultrasound scan between 2018 and 2021 at CHUSJoão (Porto) and Hospital CUF Descobertas (Lisbon), who had single pregnancy and gave informed consent. At this stage, we collected diet and lifestyle information and a random urine sample for Pb, Cd and As quantification by ICP-MS. The majority of the sample had detectable urinary levels of toxic elements (n (%) and median (P25; P75) for Pb, Cd (in ng/g), and As (in μg/ g): 980 (88) and 547.2 (289.1; 975.9), 628 (57) and 142.5 (81.5; 234.9), 1080 (97) and 27.4 (14.9; 51.3), respectively). Cd and As urinary concentrations were positively correlated (r s =0.250, p<0.001). Smoking habits were associated with higher Pb (p=0.034) and Cd (p=0.008) levels and Pb levels had a negative weak correlation with birth head circumference (r s =-0.102; p=0.009). Regarding diet, the frequencies of fish and eggs intake were positively associated with Cd and As levels (p=0.005 and p< 0.001, respectively for fish intake; p=0.030 and p<0.001, respectively, for eggs intake). Women who reported consuming fish or eggs at least four times a week presented median (P25; P75) As levels of 35.2 (21.3; 71.9) or 35.9 (16.8; 57.2) μg/g, respectively. These concentrations exceed the commonly accepted reference range (≥24.0 μg/g), observed in healthy populations. Our results evidence that, in pregnant women, smoking might be a determinant of Pb and Cd exposure and fish and eggs might be relevant determinants of Cd and As exposure. These data stress the need to study the consequences of Pb or As and Cd cumulative exposure on the health of the mother and the offspring.
- IL-10 and Cdc42 as critical modulators in methamphtamine-induced neuroinflammationPublication . Silva, Ana Isabel; Socodato, Renato; Pinto, Carolina; Terceiro, Ana Filipa; Canedo, Teresa; Relvas, João Bettencourt; Saraiva, Margarida; Summavielle, Teresa; Summavielle, TeresaPsychoactive substances, such as Methamphetamine (Meth), can induce complex neuroinflammatory responses that modulate the neuron-glia cross talk and strongly affect behavioral responses. Recently we have reported that Meth stimulates astrocytes to release tumor necrosis factor (TNF) and glutamate, leading to microglial activation, microgliosis and loss of risk-assessment. Here, we started by investigating the anti-inflammatory power the cytokine interleukin-10 (IL-10), resorting to astrocyte and microglia primary transfected with different FRET probes and exposed to Meth (100µM), to elucidate the mechanisms involved. Then after, we confirmed these results in vivo, by employing a transgenic mouse model that overexpresses IL-10 (pMT-10), in time-controlled manner, and administering a binge Meth dosing (4 x 5mg/kg, with 2h intervals). In vitro, our findings reveal that the presence of recombinant IL-10 (rIL-10) counteracts Meth-induced excessive glutamate release in astrocytes, which significantly reduced microglial activation. This reduction was associated with the modulation of astrocytic intracellular calcium (Ca2+) dynamics, particularly by restricting the release of Ca2+ from the endoplasmic reticulum to the cytoplasm. Furthermore, we identify the small Rho GTPase Cdc42 as a crucial intermediary in the astrocyte-to-microglia communication pathway under Meth. In vivo, we observed that IL-10 overexpressing prevented Meth-induced neuroinflammation, microgliosis and Meth-induced behavioral changes. These findings enhance our understanding of Meth-related neuroinflammatory mechanisms, suggesting IL-10 and Cdc42 as putative therapeutic targets, and strengthen the view of a neuroimmune nature for addiction.
- The role of RAC1 in methamphtamine-induced neuroinflammationPublication . Terceiro, Ana Filipa; Canedo, Teresa; Silva, Ana Isabel; Magalhães, Ana; Relvas, João; Summavielle, Teresa; Summavielle, TeresaMethamphetamine (Meth), a powerful psychostimulant, induces detrimental neuroinflammatory responses, in the brain reward system that seem to contribute to maintenance of addictive behaviour. Yet, the mechanisms regulating these processes in microglial cells are not clear. We have previously shown that exposing WT mice to Meth (4x5 mg/kg, 2h intervals) induces microgliosis concomitant with decreased microglia cell volume and ramification. Furthermore, psychostimulants are known to induce structural plasticity mechanisms in neurons, and Rho GTPases, important regulators of the actin cytoskeleton, are involved in these responses. Here, we assessed Rho GTPases, specifically rhoA, rac1 and cdc42, activation in response to Meth in microglia. Exposing WT mice to the same pattern of Meth administration, we found an increase in the activation of rac1 in the striatum, 15 min following the last administration of Meth. To further explore these results, we then used a conditional mice model for ablation of rac1 in adult microglia (Rac1fl/fl:Cx3cr1CreER+) and exposed these mice to the same pattern of Meth administration. Rac1 ablation was sufficient to prevent Meth-induced morphological alterations in the striatum. Currently, we are assessing the role of rac1 in the behavioural response to Meth, using a locomotor sensitization test. Overall, we identified rac1 as a novel target of Meth in microglial cells. With these results, we expect to clarify if targeting Rho GTPases may contribute to improving the treatment of addictive disorders.
- Methamphetamine activates rac1 in striatal microgliaPublication . Terceiro, Ana Filipa; Canedo, Teresa; Silva, Ana Isabel; Magalhães, Ana; Relvas, João; Summavielle, Teresa; Summavielle, TeresaMethamphetamine (Meth), a powerful psychostimulant, induces profound synaptic and morphological alterations alongside with detrimental neuroinflammatory responses, in the brain reward system. Yet, the mechanisms regulating these processes in microglial cells are not clear. We have previously shown that exposing WT mice to Meth (4x5mg/kg, 2h intervals) induces microgliosis concomitant with decreased microglia cell volume and ramification. Furthermore, psychostimulants are known to induce structural plasticity mechanisms in neurons, and Rho GTPases, important regulators of the actin cytoskeleton, are involved in these responses. Here, we evaluate if Rho GTPases, specifically rhoA, rac1 and cdc42, are critical in the response to Meth in microglia. Exposing WT mice to the samepattern of Meth administration, we found an increase in the activation of rac1 in the striatum, 15 min following the last administration of Meth. To further explore these results, we then used a conditional mice model for ablation of rac1 in adult microglia (Rac1fl/fl:Cx3cr1CreER+) and exposed these mutants to the same pattern of Meth administration. We found that rac1 ablation is sufficient to prevent Meth-induced morphological alterations in the striatum. Currently, we are assessing whether ablation of rac1 is also sufficient to prevent the neuroinflammatory response induced by Meth. Overall, we identified rac1 as a novel target of Meth in microglial cells. With these results, we expect to clarifyif targeting Rho GTPases may contribute to improving the treatment of addictive disorders.
- Microglial Rac1 is essential for microglia-synapse crosstalk and cognitive performancePublication . Almeida, Tiago O.; Socodato, Renato; Portugal, Camila C.; Santos, Evelyn C. S.; Moreira, Joana Tedim; Ferreira, João Galvão; Canedo, Teresa; Magalhães, Ana; Summavielle, Teresa; Relvas, João B.; Summavielle, TeresaMicroglia, the main immune defenders of the brain, rapidly detect and react to stimuli due to constant extension andretraction of their processes. When engaged by external cues, that can be either inflammatory or products resulting from synaptic activity, microglia dramatically change their morphology and initiate a response to reestablish brain homeostasis. Additionally, microglia can also regulate and sustain synaptic activity by secreting a plethora of factors. While some of these factors are already described, there is still much to understand on how exactly microglia-secreted factors modulate synaptic function. Rac1, a well-known member of the Rho GTPase family, is a critical regulator of actin cytoskeleton dynamics and reorganization. Furthermore, Rac1 is a component of NADPH oxidase complex, a key element for phagocytic cup formation and it also regulates NF-κB pathway activation. In the central nervous system (CNS), Rac1 is involved in axon guidance and growth, but it is also linked with Alzheimer´s disease,since it regulates the expression of amyloid precursor protein in hippocampal neurons. Although extensively studied in other cell types in and outside of the CNS, there is a profound knowledge gap on how Rac1 regulates microglia function in homeostasis and in response to external stimuli. Combining cell-specific conditional gene ablation, RNAseq, flow cytometry, immunofluorescence, proteomics and phosphoproteomics, FRET live cell imaging and mouse behavior, we aimed at describing for the first time Rac1 roles in microglial function. We observed that microglia specific Rac1 ablation impaired the capacity of microglia to sense and respond to changes in their local environment. To promote changes on the brain environment, we performed a protocol of environmental enrichment (EE), mimicking currently used therapeutic approaches for enhancing brain plasticity in patients with brain disorders. EE had a profound impact on the microglial phosphoproteomic landscape, showing a strong effect on Rho GTPase signaling. Interestingly, we showed that Rac1 signaling was the most significantly altered pathway, followed by Cdc42 and RhoA signaling, allowing us to define a hierarchy between them. Besides, EE led to an overall improvement of cognitive performance. Strikingly, ablating Rac1 from microglia completely prevented this EE-dependent cognitive enhancement and disrupted microglia-synapse crosstalk, ultimately impacting the synaptic proteomic and phosphoproteomic profiles of these mice. Overall, this is a first step into understanding how Rac1 mediates microglial responses to their local environment. This places Rac1 as anessential target for further studies and reinforces the importance of Rho GTPases signaling for adult microglial function.