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- Effect on health status and pathogen resistance of gilthead seabream (Sparus aurata) fed with diets supplemented with Gracilaria gracilisPublication . Passos, Ricardo; Correia, Ana Patrícia; Ferreira, Inês; Pires, Pedro; Pires, Damiana; Gomes, Emma; Carmo, Beatriz do; Santos, Paulo; Simões, Marco; Afonso, Clélia; Baptista, TeresaAquaculture intensification turns the reared populations prone to disease outbreaks, leading the industry to use antibiotics and chemotherapeutics. As the industry is searching for solutions, the development of sustainable aquafeeds with immunostimulant properties is emerging. This work aimed to assess the health benefits of Gracilaria gracilis as a supplement in Sparus aurata feed as well as its protective effect in a bacterial challenge. G. gracilis supplementation improved growth performance and influenced the haematological profile, namely in the constitution of the leukocytic population and erythrocytes size. Algae powder supplementation did not compromise the hepatic antioxidant systems and intestinal health and morphology were not affected by the algal supplementations. Algal extract improved lysozyme plasmatic concentration and a 5% algae powder inclusion was able to successfully protect the fish against a Photobacterium damselae subsp. piscicida infection, granting a 95% survival. In general, a 5% G. gracilis powder diet supplementation was able to improve growth, health, and bacterial resistance in gilthead seabream.
- Modulatory effect of Gracilaria gracilis on European seabass gut microbiota community and its functionalityPublication . Gonçalves, Ana Teresa; Simões, Marco; Costa, Cátia; Passos, Ricardo; Baptista, TeresaSeaweeds are an important source of nutrients and bioactive compounds and have a high potential as health boosters in aquaculture. This study evaluated the effect of dietary inclusion of Gracilaria gracilis biomass or its extract on the European seabass (Dicentrarchus labrax) gut microbial community. Juvenile fish were fed a commercial-like diet with 2.5% or 5% seaweed biomass or 0.35% seaweed extract for 47 days. The gut microbiome was assessed by 16S rRNA amplicon sequencing, and its diversity was not altered by the seaweed supplementation. However, a reduction in Proteobacteria abundance was observed. Random forest analysis highlighted the genera Photobacterium, Staphylococcus, Acinetobacter, Micrococcus and Sphingomonas, and their abundances were reduced when fish were fed diets with algae. SparCC correlation network analysis suggested several mutualistic and other antagonistic relationships that could be related to the predicted altered functions. These pathways were mainly related to the metabolism and biosynthesis of protective compounds such as ectoine and were upregulated in fish fed diets supplemented with algae. This study shows the beneficial potential of Gracilaria as a functional ingredient through the modulation of the complex microbial network towards fish health improvement.
- Identification of Gracilariaceae (Rhodophyta) of central Portugal by histological and genetic methodsPublication . Freitas, Marta V.; Simões, Marco; Valdez, Christian; Afonso, Clélia; Trindade, Beatriz G.; Correia, Ana; Baptista, Teresa; Mouga, TeresaThis study aims to identify different populations of Gracilariaceae collected from the central coast of Portugal through light microscopy, anatomical observations and genetic tools, essential approaches to correctly assign species identity. Samples were obtained from Ria de Aveiro (AV), Figueira da Foz (FFBC, FFMD), and Lagoa de Óbidos (LOBR, LOEV, LOBS). Although histological observations offered a visual representation of the characteristic pseudoparenchymatous organization, they did not allow a clear distinction among the species. The amplification of a ∼700 bp fragment of the mitochondrial cytochrome oxidase I gene, and its sequencing enabled us to assign the populations FFBC and LOBS to Gracilaria gracilis, and the populations AV, FFMD, LOBR, and LOEV to Agarophyton vermiculophyllum. This contribution will help phycologists to correctly identify the Portuguese populations of Gracilaria sensu lato at the species level, which will be crucial in ensuring that future studies and industrial exploration accurately target the correct species.
- Crosstalk between energy and gibberellin signaling pathways: a molecular approachPublication . Simões, Marco Aurélio Dias; Rodrigues, Américo do PatrocínioAs plantas, como todos os organismos, têm de enfrentar uma grande variedade de stresses bióticos e abióticos, mas sendo organismos sésseis elas não são capazes de escapar a estas condições adversas. Portanto, a sua sobrevivência é criticamente depende da sua resposta eficiente às mudanças ambientais. Direta ou indiretamente, o stress geralmente leva a um défice de energia. Se a homeostasia energética não for restabelecida, o crescimento e o desenvolvimento da planta são drasticamente comprometidos. SNF1 (sucrose non-fermenting 1) -related protein kinase 1.1 (SnRK1.1) desempenha um papel importante na coordenação das respostas de energia e stress. Esta kinase deteta e sinaliza condições de stress, tais como escuridão inesperada, baixo teor de açúcar e outros stresses, atuando em genes que controlam uma rede de genes de transcrição. Nas últimas décadas, vários estudos têm revelado um cruzamento entre as vias de sinalização do açúcar e das hormonas. Um estudo recente identificou as proteínas DELLA como um interator de SnRK1 na presença de NDF4, utilizando o sistema Y2H. As proteínas DELLA são reguladoras negativas da via de sinalização da giberelina. As giberelinas são hormonas vegetais que controlam vários processos de desenvolvimento, como a germinação e o alongamento do caule. Neste trabalho, nós confirmámos in vivo e in vitro a interação entre SnRK1.1 e as proteínas DELLA e demonstrámos que esta interação é independente da presença de NDF4. As análises fenotípicas de mutantes sobre-expressores de SnRK1.1 revelaram que este mutante é insensível a giberelinas e hipersensível a paclobrutazol. Estes resultados podem sugerir que SnRK1 interage e fosforila as proteínas DELLA e que esta fosforilação aumenta a sua estabilidade e consequentemente diminui a resposta a giberelinas.
- Potential use of macroalgae Gracilaria gracilis in diets for European seabass (Dicentrarchus labrax): Health benefits from a sustainable sourcePublication . Passos, Ricardo; Correia, Ana Patrícia; Pires, Damiana; Pires, Pedro; Ferreira, Inês; Simões, Marco; Carmo, Beatriz do; Santos, Paulo; Pombo, Ana; Afonso, Clélia; Baptista, TeresaSeaweeds still possess a large undisclosed potential, mainly due to their constituent’s richness, which may have several uses for society. In aquaculture, they may play a role as an ecological sustainable aquafeed supplement to increase overall health and fight pathogenic outbreaks. This study aimed to evaluate the general health modulation that the inclusion of Gracilaria gracilis could accomplish in the diet of Dicentrarchus labrax. Dried algae at 2.5% and 5% and algal extract at 0.35% inclusion levels were supplemented to seabass diet to evaluate possible growth, haematological, immunological, antioxidant, metabolic, and intestinal morphological modulations. The supplementations did not impact growth or feed utilization, and barely affected the haematological profile and some metabolic parameters. Nevertheless, it caused a marked outcome on lysozyme, some oxidative stress biomarkers, and intestine morphology, suggesting beneficial consequences from the algal inclusion. Dried algae powder, with a 2.5% inclusion, boosted immune response, with higher plasmatic lysozyme and intestinal acid goblet cells and protected against oxidative damages by improved enzymatic and non-enzymatic responses. Thus, we provide evidence that dietary seaweed application may be a path towards a more sustainable aquaculture industry.
- Early immune modulation in European Seabass (Dicentrarchus labra) juveniles in response to Betanodavirus infectionPublication . Vaz, Mariana; Pires, Damiana; Pires, Pedro; Simões, Marco; Pombo, Ana; Santos, Paulo; Carmo, Beatriz do; Passos, Ricardo; Costa, Janina Z.; Thompson, Kim D.; Baptista, TeresaThe early host–pathogen interaction between European seabass (Dicentrarchus labrax) and Betanodavirus was examined by using juvenile fish infected intramuscularly with RGNNV (red-spotted grouper nervous necrosis virus). The time course selected for sampling (0–144 h post-infection (hpi)) covered the early stages of infection, with hematological, antioxidant and immunological responses examined. Early activation of the host’s immune system was seen in the first few hours post-infection (6 to 9 hpi), as evidenced by an increase in tnfa, cd28 and c3 expression in the head kidney of infected fish. Most hematological parameters that were examined showed significant differences between sampling times, including differences in the number of thrombocytes and various leukocyte populations. The plasma lysozyme concentration decreased significantly over the course of the trial, and most antioxidant parameters examined in the liver showed significant differences over the infection period. At 144 hpi, peak expression of tnfa and il-1b coincided with the appearance of disease symptoms, peak levels of virus in the brain and high levels of fish mortality. The results of the study show the importance of analyzing the early interactions between European seabass and Betanodavirus to establish early indicators of infection to prevent more severe outcomes of the infection from occurring.
- Short-term immune responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicidaPublication . Santos, Paulo; Peixoto, Diogo; Ferreira, Inês; Passos, Ricardo; Pires, Pedro; Simões, Marco; Pousão-Ferreira, Pedro; Baptista, Teresa; Costas, BenjaminPhotobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 uL of PBS (placebo) or 100 uL of exponentially growing Phdp (1 x 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.
- Disclosing the potential of eleganolone for Parkinson’s disease therapeutics: neuroprotective and anti-inflammatory activitiesPublication . Silva, Joana; Alves, Celso; Pinteus, Susete; Susano, Patrícia; Simões, Marco; Guedes, Miguel; Martins, Alice; Rehfeldt, Stephanie; Gaspar, Helena; Goettert, Márcia Inês; Alfonso, Amparo; Pedrosa, RuiThe treatment of Parkinson´s disease (PD) has benefited from significant advances resulting from the increasing research efforts focused on new therapeutics. However, the current treatments for PD are mostly symptomatic, alleviating disease symptoms without reversing or retarding disease progression. Thus, it is critical to find new molecules that can result in more effective treatments. Within this framework, this study aims to evaluate the neuroprotective and anti-inflammatory effects of three compounds (eleganolone, eleganonal and fucosterol) isolated from the brown seaweed Bifurcaria bifurcata. In vitro neuroprotective effects were evaluated on a PD cellular model induced by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y human cells, while lipopolysaccharide (LPS) -stimulated RAW 264.7 macrophages were used to evaluate the anti-inflammatory potential. Additionally, the underlying mechanisms of action were also investigated. Compounds were isolated by preparative chromatographic methods and their structural elucidation attained by NMR spectroscopy. Among the tested compounds, eleganolone (0.1–1 μM; 24 h) reverted the neurotoxicity induced by 6-OHDA in about 20%. The neuroprotective effects were mediated by mitochondrial protection, reduction of oxidative stress, inflammation and apoptosis, and inhibition of NF-kB pathway. The results suggest that eleganolone may provide advantages in the treatment of neurodegenerative conditions and, therefore, should be considered for future preclinical studies.
- RGA & GAI: On the characterization of two DELLA plant growth repressor proteinsPublication . Vaz, Daniela C.; Sousa, Raquel; Simões, Marco; Rodrigues, AméricoDELLA proteins are a family of nuclear proteins responsible for plant growth modulation. They act as growth repressor proteins in response to gibberellin signaling pathways. Five DELLA protein homologs were found in Arabidopsis thaliana, namely, RGA (Repressor of Gibberellic Acid), GAI (Gibberellic Acid Insensitive) and three RGA-like proteins (RGL-1, RGL-2 and RGL-3). The RGA- DELLA and GAI-DELLA protein homologs have been classified as Intrinsically Unstructured Proteins (IUPs) that undergo a disorder-to-order transition upon receptor binding. This structural change has found to be physiologically relevant for biological signaling and molecular recognition. Thus, in order to better characterize the structural features and molecular changes that govern these conformational variations of the polypeptide chain, we have produced recombinant RGADELLA and GAI-DELLA proteins in three length-versions, i.e. full-length, N-terminal and C-terminal versions. Full-length and terminal versions present different sequence motifs, attributed to different biological functions. All proteins were analyzed spectro-photometrically, via Light Scattering (LS), Circular Dichroism (CD) and intrinsic and extrinsic fluorescence (ANS binding), in order to compare spectral profiles, secondary structure propensities, levels of solvent exposure and structural compactness. Full-length and terminal variants exhibit different behaviors, spectral profiles and levels of compactness that can be related to different protein domains and ultimately to different functional implications.
- Detection of mcr-1 Gene in undefined Vibrio species isolated from clamsPublication . Valdez, Christian; Costa, Cátia; Simões, Marco; Carvalho, Carla C. C. R. De; Baptista, Teresa; Campos, MariaThe increase of antimicrobial resistant strains is leading to an emerging threat to public health. Pathogenic Vibrio are responsible for human and animal illness. The Enterobacteriaceae family includes microorganisms that affect humans, causing several infections. One of the main causes of human infection is related to the ingestion of undercooked seafood. Due to their filterfeeding habit, marine invertebrates, such as clams, are known to be a natural reservoir of specific microbial communities. In the present study, Vibrionaceae and coliforms microorganisms were isolated from clams. A microbial susceptibility test was performed using the disk diffusion method. From 43 presumptive Vibrio spp. and 17 coliforms, three Vibrio spp. with MICs to colistin > 512 mg L−1 were found. From the 23 antimicrobial resistance genes investigated, only the three isolates that showed phenotypic resistance to colistin contained the mcr‐1 gene. Genotypic analysis for virulence genes in EB07V indicated chiA gene presence. The results from the plasmid cure and transformation showed that the resistance is chromosomally mediated. Biochemical analysis and MLSA, on the basis of four protein‐coding gene sequences (recA, rpoB, groEL and dnaJ), grouped the isolates into the genus Vibrio but distinguished them as different from any known Vibrio spp.