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- Sustainable carotenoid extraction from macroalgae: Optimizing microwave-assisted extraction using response surface methodologyPublication . Lopes, Andreia; Correia-Sá, Luísa; Vieira, Mónica; Delerue-Matos, Cristina; Soares, Cristina; Grosso, Clara; Vieira, MónicaThis study aimed at optimizing carotenoid extraction using the macroalga Himanthalia elongata (L.) S.F.Gray as a model. Firstly, traditional extraction procedures were employed, using various solvents and temperatures to enhance the extraction conditions. Once the most effective extraction conditions were identified, the study transitioned to a more efficient and environmentally friendly approach, microwave-assisted extraction (MAE). By applying a three-parameter (solid-tosolvent ratio, temperature, and time) Box–Behnken design, the optimal extraction conditions were found to be a solid-to-solvent ratio of 1/13.6 g/mL at 60 ◦C for 15 min. Under these conditions, the predicted and experimental carotenoid contents were 2.94 and 2.12 µg/mL, respectively. Furthermore, an HPLC-DAD method was developed and validated for the characterization of carotenoids. βCarotene was the predominant carotenoid in H. elongata, alongside fucoxanthin. The optimized MAE method was applied to other seaweeds, including Fucus vesiculosus L., Codium tomentosum Stackhouse, Gracilaria gracilis (Stackhouse) Steentoft, L.M.Irvine & Farnham, and Eiseinia bicyclis (Kjellman) Setchell. Among all, F. vesiculosus exhibited the highest carotenoid content compared to the others. This study concludes that MAE under optimized conditions is an effective and sustainable approach for carotenoid extraction, providing significant yields of bioactive compounds such as β-carotene and fucoxanthin, which have promising applications in enhancing human health and nutrition.
- High efficacy of chloroquine-derived bile salts in Pluronic F127 micelles against blood-stage Plasmodium falciparumPublication . Silva, Ana Teresa; Prudêncio, Miguel; Oliveira, Isabel S.; Nogueira, Fátima; Morais, Inês; Santana, Sofia; Ferraz, Ricardo; Workneh, Eyob A.; Gomes, Paula; Marques, Eduardo F.; Ferraz, RicardoColloidal nanocarriers can play a key role in the efficacious delivery of drugs, including antimalarials. Here, we investigated the ability of polymeric micelles of the block copolymer F127 to act as nanovehicles for two organic salts derived from chloroquine and human bile acids, namely, chloroquinium cholate (iCQP1) and chloroquinium glycocholate (iCQP1g). We have previously reported the strong in vitro antiplasmodial activity of these salts, which displayed IC50 values of 13 and 15 nM against blood forms of Plasmodium falciparum, respectively. By deriving from amphiphilic lipids, iCQP1 and iCQP1g also enclose the ability to act as surface-active ionic liquids (SAILs). The micellization properties of neat F127 and of the F127/SAIL mixtures were initially investigated to gain physicochemical insight into the interaction between polymer and bioactive SAILs, resorting to differential scanning calorimetry, surface tension measurements and dynamic light scattering. Micelle formation by F127 is an endothermic process strongly temperature and concentration dependent. Interestingly, this process is significantly changed when the molar fraction of SAIL (xSAIL) in the F127/SAIL mixture is varied between 0.33 and 0.90. Both SAILs favor the formation of mixed micelles by decreasing the micellization temperature, and (observed only when for xSAIL = 0.33) by synergistically decreasing the cmc. Concomitantly, the micellar size is reduced from 18 to 13 nm as xSAIL is increased from 0.33 to 0.90. Crucially, in vitro assays show that when the SAILs are loaded into F127 polymeric micelles, their antiplasmodial efficacy is substantially enhanced, with a significant drop in IC50, especially for the iCQP1/F127 system. This opens new possibilities for the nanoformulations of antimalarial compounds.
- Effect of competitive exclusion in rabbits using an autochthonous probioticPublication . Cunha, Sara; Mendes, Ângelo; Rego, Dinis; Meireles, Diana; Fernandes, Ruben; Carvalho, André; Costa, Paulo Martins da; Fernandes, RúbenAnimal nutrition has been severely challenged by the ban on antimicrobials as growth promoters. This has fostered the study of alternative methods to avoid colonisation by pathogenic bacteria as well as to improve the growth of animals and feed conversion efficiency. These new options should not alter the normal intestinal microbiota, or affect it as little as possible. The use of probiotics, which are live microorganisms that beneficially affect the host by improving its intestinal microbial balance, can be seen as a promising way to achieve that goal. In this study, New Zealand White rabbits were fed diets containing an autochthonous probiotic of Enterococcus spp., with the strains EaI, EfaI and EfaD, and Escherichia coli, with the strains ECI 1, ECI 2 and ECD, during a 25-d trial, to evaluate the impact of the probiotic on the faecal microbiota, including population dynamics and antimicrobial resistance profiles. A control group of rabbits, which was fed a diet containing a commonly used mixture of antimicrobials (colistin, oxytetracycline, and valnemulin), was also studied. To assess the colonisation ability of the mentioned probiotic, the faecal microbiota of the rabbits was characterised up to 10 d after the administration had ended. Isolates of enterococci and E. coli were studied for phylogenetic relationships using enterobacterial repetitive intergenic consensus (ERIC-PCR) and pulse-field gel electrophoresis (PFGE), respectively. Although partially affected by an unexpected clinical impairment suffered by the rabbits in the experimental group, our results showed the following. The difference between the growth rate of the animals treated with antimicrobials and those fed the probiotic was not statistically significant (P> 0.05). The competitive exclusion product was present in the faecal samples in a large proportion, but stopped being recovered by culture as soon as the administration ended and the housing conditions were changed. Multidrug-resistant strains of enterococci and E. coli were more commonly recovered from faecal samples of animals fed diets containing antimicrobials, than from rabbits fed diets with our probiotic formula. The use of E. coli probiotics to prevent infection by enteropathogenic strains must be carefully considered due to the possible occurrence of gastrointestinal signs. On the other hand, enterococci strains may be more effective, but lack the long-term colonisation ability.
- Molecular characterization of quinolone resistance mechanisms and extended-spectrum ß-lactamase production in Escherichia coli isolated from dogsPublication . Meireles, D.; Leite-Martins, L.; Bessa, J.; Cunha, S.; Fernandes, Ruben; Matos, A. de; Manaia, C.M.; Costa, P. Martins da; Fernandes, RúbenThe increasing prevalence of antimicrobial resistances is now a worldwide problem. Investigating the mechanisms by which pets harboring resistant strains may receive and/or transfer resistance determinants is essential to better understanding how owners and pets can interact safely. Here, we characterized the genetic determinants conferring resistance to β-lactams and quinolones in 38 multidrug-resistant Escherichia coli isolated from fecal samples of dogs, through PCR and sequencing. The most frequent genotype included the β-lactamase groups TEM (n = 5), and both TEM + CTX-M-1 (n = 5). Within the CTX-M group, we identified the genes CTX-M-32, CTX-M-1, CTX-M-15, CTX-M-55/79, CTX-M-14 and CTX-M-2/44. Thirty isolates resistant to ciprofloxacin presented two mutations in the gyrA gene and one or two mutations in the parC gene. A mutation in gyrA (reported here for the first time), due to a transversion and transition (TCG → GTG) originating a substitution of a serine by a valine in position 83 was also detected. The plasmid-encoded quinolone resistance gene, qnrs1, was detected in three isolates. Dogs can be a reservoir of genetic determinants conferring antimicrobial resistance and thus may play an important role in the spread of antimicrobial resistance to humans and other co-habitant animals.
- High resistance to fourth-generation cephalosporins among clinical isolates of Enterobacteriaceae producing extended-spectrum β-lactamases isolated in PortugalPublication . Prudêncio, Cristina; Gestoso, Álvaro; Freitas, José Mota; Santos, Perpétua; Prudêncio, Cristina; Fernandes, Ruben; Fernandes, RúbenHere we report the molecular and antimicrobial susceptibility profile of extended-spectrum β-lactamase (ESBL)-producing strains found in the Portuguese northern occidental coast region (Minho). For this purpose, bacteria isolated from clinical hospitalised and non-hospitalised patients over a period of 2 years were identified and minimal inhibitory concentrations (MICs) were determined by microdilution methods according to the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) guidelines on Enterobacteriaceae. Additionally, ESBL phenotypic identification was confirmed by the Etest (AB BIODISK, Solna, Sweden). Various methods of molecular identification of the β-lactamase (bla) genes, involving polymerase chain reaction (PCR) and sequencing strategies, were used in this study. The ESBL-producing strains (n = 193) were isolated from urine (n = 127), sputum (n = 42), bronchoalveolar lavage (n = 14), blood (n = 7) and ascitic fluid (n = 3). The most frequent ESBL-producing organism isolated in the present study was Escherichia coli (67.9%; n = 131), followed by Klebsiella pneumoniae (30.6%; n = 59), Klebsiella oxytoca (0.5%; n = 1), Enterobacter aerogenes (0.5%; n = 1) and Citrobacter freundii (0.5%; n = 1). The ESBL detected in the present studywere the TEM type (40.4%), CTX-M type (36.8%) and SHV type (22.8%). TEM-52 and TEM-24 were the most frequent TEM types (20.2% and 12.9%, respectively). Members of TEM-10 (4.1%) and TEM-116 (2.1%) were also detected. Within the CTX-M family, CTX-M-9 group was represented by CTX-M-9 (13.5%) and CTX-M-14 (8.4%). In the CTX-M-1 group, CTXM-15 was the most frequent type (12.4 %), followed by CTX-M-1 (2.1%), CTX-M-3 (0.5%) and CTX-M-32 (0.5%). Regarding CTX-M types, it appears that CTX-M-14 is widespread among the northwestern Iberian Peninsula [1]. Klebsiella pneumoniae harbouring a CTX-M-15 enzyme was described for the first time in Portugal in 2005 [2] in the Lisbon area, but CTX-M-15 enzyme has also recently been found by us in the north of Portugal in another Enterobacteriaceae member, isolated from bloodstream infections [3] among seven patients in two different hospitals. Other ESBL-producing species (not E. coli or K. pneumoniae)were also found. This is the first time that C. freundii has been described as a producer of CTX-M-32 in this country. The SHV enzymes occurred only in 23.3% of all ESBL-producing organisms. Within this type, the most frequent type was SHV-12 (12.4%), followed by SHV-5 (8.8%) and finally SHV-2 (2.1%). Some isolates co-produced more than one ESBL type: TEM-52/CTX-M-14 (0.5%); TEM-116/CTX-M-14 (0.5%); and TEM-116/CTX-M-15 (0.5%). MIC testing showed that isolates producing ESBLs were mostly susceptible to carbapenems (100%) and amikacin (99.5%). In contrast, ESBL-producing strains presented low susceptibility rates to cefepime and quinolones. Indeed, 98.9% of the ESBL-producing strains were cefepime-resistant and 85.4% were resistant to quinolones (ciprofloxacin and norfloxacin). In the generality, these high levels of resistance to quinolones were more conspicuous in members of the CTX-M family (98.1%) than TEM and SHV types (80.8% and 72.1%, respectively). In this study, cefepime presented a surprisingly low activity against ESBL-producing microorganisms. Recent literature refers to the inoculum effect exhibited by cefepime [4]. Nevertheless, we believe that this should not be pointed out as a single explanation once MIC determination is performed using inoculum concentrations of 0.5 McFarland standard. In our sample, only two K. pneumoniae harbouring SHV-2 ESBL were susceptible to cefepime. All the other clinical isolates (98.9%) expressing the ESBL phenotype were resistant to cefepime. It seems interesting that a recent study showed that cefepimewas successfully administered to three patients (two females and one male) aged between 47 years and 87 years carrying a Gram-negative ESBL-positive strain [5]. Nevertheless, other studies worldwide have begun to describe the emergence of high resistance to cefepime among Gram-negative ESBL-producers [6]. The present work showed a high diversity of ESBL enzymes occurring in the north of Portugal. In this country, the most prevalent type is still the TEM type, but CTX-M is growing rapidly [7]. The emergence of ESBL-producers resistant to cefepime in Portugal is a matter of concern. We believe that the uncontrolled use of cephalosporins may have an important role in the acquisition of resistance mechanisms, particularly the production of ESBL enzymes. Establishment of policies to monitor drug delivery in hospital and ambulatory pharmacies as well as implementation of public health defence strategies towards health promotion and drug resistance prevention appear to be urgent.
- Pericardial fluid accumulates microRNAs that regulate heart fibrosis after myocardial infarctionPublication . Silva, Elsa D.; Pereira-Sousa, Daniel; Ribeiro-Costa, Francisco; Cerqueira, Rui; Enguita, Francisco J.; Gomes, Rita N.; Dias-Ferreira, João; Pereira, Cassilda; Castanheira, Ana; Pinto-do-Ó, Perpétua; Leite-Moreira, Adelino F.; Nascimento, Diana S.; Lopes Pereira, Cassilda MariaPericardial fluid (PF) has been suggested as a reservoir of molecular targets that can be modulated for efficient repair after myocardial infarction (MI). Here, we set out to address the content of this biofluid after MI, namely in terms of microRNAs (miRs) that are important modulators of the cardiac pathological response. PF was collected during coronary artery bypass grafting (CABG) from two MI cohorts, patients with non-ST-segment elevation MI (NSTEMI) and patients with ST-segment elevation MI (STEMI), and a control group composed of patients with stable angina and without previous history of MI. The PF miR content was analyzed by small RNA sequencing, and its biological effect was assessed on human cardiac fibroblasts. PF accumulates fibrotic and inflammatory molecules in STEMI patients, namely causing the soluble suppression of tumorigenicity 2 (ST-2), which inversely correlates with the left ventricle ejection fraction. Although the PF of the three patient groups induce similar levels of fibroblast-to-myofibroblast activation in vitro, RNA sequencing revealed that PF from STEMI patients is particularly enriched not only in pro-fibrotic miRs but also anti-fibrotic miRs. Among those, miR-22-3p was herein found to inhibit TGF-β-induced human cardiac fibroblast activation in vitro. PF constitutes an attractive source for screening diagnostic/prognostic miRs and for unveiling novel therapeutic targets in cardiac fibrosis
- Investigating multiple vegetable oils and recycled variant for microplastics extraction from water, integrated with Raman spectroscopyPublication . Garzón-Vidueira, R.; André, M.; Rede, D.; Barbosa, R.; Rial-Otero, R.; Delerue-Matos, C.; Vilarinho, Rui; Fernandes, Vírginia Cruz; Vilarinho, RuiThe global production and disposal of plastics have led to pervasive contamination of natural environments, representing considerable risks to human health and ecosystems. This study introduces a novel oil-based method for extracting microplastics (MPs) from water samples, with a focus on optimizing extraction conditions and improving the quality of MPs identification using Raman spectroscopy. Various parameters including the type of oil, salinity, temperature, air incorporation, and washing solvent were investigated to enhance extraction efficiency and spectroscopic identification accuracy. Sunflower oil emerged as the preferred extraction medium due to its compatibility with Raman spectroscopy, offering high recovery efficiencies for polypropylene (PP) and polystyrene (PS). Additionally, ethanol was identified as a better washing solvent compared to hexane, improving MPs identification. The optimised method was then applied to environmental water samples, revealing matrix effects and challenges with digestion step. Despite these challenges, the proposed method represents a significant advancement in microplastic analysis, offering reliable detection and quantification in aquatic environments. Further optimisation is needed to address matrix effects and improve recovery efficiency, especially for smaller microplastics.
- Micro(nano)plastic and related chemicals: Emerging contaminants in environment, food and health impactsPublication . Carvalho, Juliana G. R. de; Augusto, Helga Coelho; Ferraz, Ricardo; Delerue-Matos, Cristina; Fernandes, Virgínia Cruz; Fernandes, Virgínia; Ferraz, RicardoMicroplastic pollution is a problem of increasing concern in food, and while food safety issues around the world are serious, an increasing number of food safety issues related to microplastics have become the focus of people’s attention. The presence of microplastics in food is a worldwide problem, and they are present in all kinds of foods, foods of both animal and plant origin, food additives, drinks, plastic food packaging, and agricultural practices. This can cause problems for both humans and the environment. Microplastics have already been detected in human blood, heart, placenta, and breastmilk, but their effects in humans are not well understood. Studies with mammals and human cells or organoids have given perspective about the potential impact of micro(nano)plastics on human health, which affect the lungs, kidneys, heart, neurological system, and DNA. Additionally, as plastics often contain additives or other substances, the potentially harmful effects of exposure to these substances must also be carefully studied before any conclusions can be drawn. The study of microplastics is very complex as there are many factors to account for, such as differences in particle sizes, constituents, shapes, additives, contaminants, concentrations, etc. This review summarizes the more recent research on the presence of microplastic and other plastic-related chemical pollutants in food and their potential impacts on human health.
- Bioactive peptides from milk proteins with antioxidant, anti-inflammatory, and antihypertensive activitiesPublication . Borges, Thaís; Coelho, Pedro; Prudêncio, Cristina; Gomes, Ana; Gomes, Paula; Ferraz, Ricardo; Coelho, Pedro; Prudêncio, Cristina; Ferraz, RicardoPeptides from protein ingredients exhibit key biological activities, including antimicrobial, antihypertensive, antioxidant, anti-inflammatory, analgesic, and immunomodulatory effects. Aligning with the One Health approach, there is growing investment in promoting pet health and well-being. As a result, sustainable functional ingredients are increasingly essential for pet food development. In this work, peptides derived from lactoferrins of different mammalian species were synthesized and their antioxidant, anti-inflammatory, and antihypertensive activities were investigated. This study examined the antioxidant, anti-inflammatory, antihypertensive activities, and cytotoxicity of bioactive peptides derived from lactoferrins of various mammalian species through spectroscopical methods. The peptides were produced via chemical synthesis (bottom-up approach). Peptides derived from bovine lactoferrin showed the most promising antioxidant and anti-inflammatory activities, whereas those derived from human lactoferrin showed the highest antihypertensive effects and the lowest cytotoxicity. In short, milk-derived peptides with antioxidant, anti-inflammatory, and antihypertensive activity were identified. This motivates further studies to better characterize these peptides, including their properties and pharmacokinetics in vivo, to assess their true potential as nutraceutical agents.
- Screening for microplastics in agricultural soils: Applying green chemistry principles in extraction and analysisPublication . Rede, Diana; Vilarinho, Rui; Delerue-Matos, Cristina; Moreira, Joaquim Agostinho; Fernandes, Virgínia Cruz; Fernandes, VirgíniaIn recent years, microplastic (MP) pollution has garnered significant attention owing to its ability to permeate various ecosystems, including soil. These particles can infiltrate the environment, either directly or through the degradation of larger plastic items. Despite growing concerns, standardized methods for quantification are still lacking. This study aimed to screen for the presence of MPs in agricultural soils while incorporating green analytical principles in the methodology. A density separation followed by centrifugation was employed, based on the principles of the QuEChERS extraction method. This approach minimized sample quantities, reagent consumption, and waste production, ensuring efficient extraction and analysis. Recovery tests using certified soils spiked with pristine MPs, specifically polystyrene, polypropylene (PP), and ethylene-vinyl acetate for larger MPs (3–5 mm), and low-density polyethylene, polyamide 6, and tire wear particles for smaller MPs (15–300 m), achieved recovery levels exceeding 69% for smaller MPs and over 91% for larger particles. Spectroscopic analysis revealed slight alterations in the Raman spectra of MPs after extraction. Transitioning to agricultural soil analysis has revealed challenges, including spectral interferences. Nine mesoplastics (5–20 mm) were detected, predominantly consisting of PP and polyethylene (PE), along with seven MPs, three of which were individually identified as PE-based, while the remainder were inconclusive, including one fiber. The evaluation of the method’s sustainability using the Analytical Eco-Scale and Analytical Greenness Calculator Metric (AGREE), with scores of 82 out of 100 and 0.66 out of 1, respectively, demonstrated its potential as a reliable approach to MP analysis in soils. This study highlights the potential of integrating green analytical chemistry principles into MP extraction methodologies and emphasizes the value of the proposed QuEChERs-based approach for improving the sustainability and efficiency of MP monitoring in agricultural soils.