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- C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in ArabidopsisPublication . Rodriguez, Lesia; Gonzalez-Guzman, Miguel; Diaz, Maira; Rodrigues, Américo; Izquierdo-Garcia, Ana C.; Peirats-Llobet, Marta; Fernandez, Maria A.; Antoni, Regina; Fernandez, Daniel; Marquez, Jose A.; Mulet, Jose M.; Albert, Armando; Rodriguez, Pedro L.Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, andCAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.
- Carica papaya latex: A low‐cost biocatalyst for human milk fat substitutes productionPublication . Tecelão, Carla; Rivera, Ivanna; Sandoval, Georgina; Ferreira‐Dias, SuzanaAbstractThis work aims at evaluating the potential of Carica papaya lipase (CPL) self‐immobilized in papaya latex as a biocatalyst for the synthesis of human milk fat substitutes (HMFS), to be used as a low‐cost alternative to commercial lipases. Two different CPL preparations, one extracted from the papaya fruit (CPL I) and the other from petiole leaves (CPL II) of papaya tree, were tested as catalysts for the acidolysis between tripalmitin and (i) oleic acid or (ii) omega‐3 PUFA, batchwise, at 60°C, in solvent‐free media. After 24 h, molar incorporation was higher for oleic acid (22.1 mol%) when CPL I was used. This biocatalyst was selected for further studies. RSM was used to model reaction conditions: medium formulation (molar ratio oleic acid/tripalmitin, MR, 1.2:1–6.8:1) and temperature (58–72°C). Acyl migration decreased with MR increase. In batch operational stability assays at 60°C, using MR of 2:1 and 6:1, the highest stability was observed for a MR of 2:1.Practical applications: The use of this biocatalyst is a feasible way to valorize papaya agro‐residues which represent an important environmental problem in the producing countries. The obtained results were rather promising since, with this almost zero‐cost biocatalyst, it was possible to produce a high added‐value product (HMFS). Under optimized conditions, the obtained results were comparable with those obtained with expensive immobilized commercial lipases.
- Cholinesterase activity in the caddisfly Sericostoma vittatum: Biochemical enzyme characterization and in vitro effects of insecticides and psychiatric drugsPublication . Pestana, João L. T.; Novais, Sara C.; Lemos, Marco; Soares, Amadeu M. V. M.Sericostoma vittatum is a caddisfly species, endemic to the Iberian Peninsula, proposed as a biomonitor species for lotic ecosystems. Since inhibition of cholinesterases[U+05F3] (ChE) activity has been used to evaluate the exposure of macroinvertebrates to organophosphates and carbamate pesticides, this work intended to characterize the ChE present in this species so their activity can be used as a potential biomarker of exposure. Biochemical and pharmacological properties of ChE were characterized in this caddisfly species using different substrates (acetylthiocholine iodide, propionylthiocholine iodide, and butyrylthiocholine iodide) and selective inhibitors (eserine sulfate, BW284c51, and iso-OMPA). Also, the in vitro effects of two insecticides (carbaryl and chlorantraniliprole) and two psychiatric drugs (fluoxetine and carbamazepine) on ChE activity were investigated. The results suggest that S. vittatum possess mainly AChE able to hydrolyze both substrates acetylthiocholine and propionylthiocholine since: (1) it hydrolyzes the substrate acetylthiocholine and propionylcholine at similar rates and butyrylthiocholine at a much lower rate; (2) it is highly sensitive to eserine sulfate and BW284c51, but not to iso-OMPA; and (3) its activity is inhibited by excess of substrate, a characteristic of typical AChE. in vitro inhibitions were observed only for carbaryl exposure while exposure to chlorantraniliprole and to relevant environmental concentrations of psychiatric drugs did not cause any significant effect on AChE activity. This study suggests that AChE activity in caddisflies can indeed be used to discriminate the effects of specific insecticides in monitoring programs. The use of non-target species such as caddisflies in ecotoxicological research in lotic ecosystems is also discussed.
- Detection of QnrB54 and Its Novel Genetic Context in Citrobacter freundii Isolated from a Clinical CasePublication . Campos, Maria Jorge; Palomo, Gonzalo; Hormeño, Lorena; Rodrigues, Américo; Sánchez-Benito, Rosario; Píriz, Segundo; Quesada, AlbertoLETTER Quinolone resistance in Enterobacteriaceae is mediated by mutations in the quinolone resistance-determining regions (QRDR) of topoisomerase genes and/or by plasmid-mediated quinolone resistance determinants (PMQR) such as the qnr genes encoding pentapeptide repeat proteins (1). The qnrB family is represented by 80 different alleles (http://www.lahey.org/qnrStudies/); most of them originated from Citrobacter strains and spread to other Enterobacteriaceae species (2). This work describes the identification of a new allele of the quinolone resistance protein QnrB, QnrB54, in a human clinical isolate of Citrobacter freundii detected in Spain.
- Developmental Toxicity of Endocrine Disrupters Bisphenol A and Vinclozolin in a Terrestrial IsopodPublication . Lemos M.F.L.; van Gestel, C. A. M.; Soares, A. M. V. M.Studies of the effects of endocrine-disrupting compounds (EDCs) on invertebrates are still largely underrepresented. This work aims to fill this gap by assessing the effects of bisphenol A (BPA) and vinclozolin (Vz) on the terrestrial isopod Porcellio scaber (common rough woodlouse). Male adult and sexually undifferentiated juvenile woodlice were exposed to the toxicants. Effects on molting regime and growth were investigated independently for males and female woodlice after sexual differentiation. Both chemicals elicited developmental toxicity to P. scaber by causing overall decreased growth. Nevertheless, BPA induced molting, whereas Vz delayed it. Although the LC 50 values for juvenile and adult survival were fairly similar, juvenile woodlice showed an increased chronic sensitivity to both chemicals, and female woodlice were most the sensitive to BPA. We recommend the use of adults, juveniles, female, and male woodlice, as well as a large range of toxicant concentrations, to provide valuable information regarding differential dose responses, effects, and threshold values for EDCs.
- Ecotoxicological Effects of the Anionic Surfactant Sodium Dodecyl Sulfate (SDS) in Two Marine Primary Producers: Phaeodactylum tricornutum and Ulva lactucaPublication . Cruz de Carvalho, Ricardo; Feijão, Eduardo; Matos, Ana Rita; Cabrita, Maria Teresa; Utkin, Andrei B.; Novais, Sara C.; Lemos, Marco F. L.; Caçador, Isabel; Marques, João Carlos; Reis-Santos, Patrick; Fonseca, Vanessa F.; Duarte, BernardoSodium Dodecyl Sulfate (SDS) is an anionic surfactant, extensively used in detergents, household and personal care products, as well as in industrial processes. The present study aimed to disclose the potential toxicological effects of SDS exposure under environmentally relevant concentrations (0, 0.1, 1, 3, and 10 mg L−1) on the physiology and biochemistry (photosynthesis, pigment, and lipid composition, antioxidative systems, and energy balance) of two marine autotrophs: the diatom Phaeodactylum tricornutum and the macroalgae Ulva lactuca. A growth rate (GR) reduction in P. tricornutum was observed with a classic dose-response effect towards the highest applied concentration, while a GR increase occurred in U. lactuca. Regarding photochemistry, the decrease in the fluorescence of the OJIP curves and laser-induced fluorescence allowed a better separation between SDS treatments in U. lactuca compared with P. tricornutum. Although all pigments significantly decreased in U. lactuca at the highest concentrations (except for antheraxanthin), no significant variations occurred in P. tricornutum. On the other hand, changes in fatty acid content were observed in P. tricornutum but not in U. lactuca. In terms of classical biomarker assessment, a dose-effect relationship of individual biomarkers versus SDS dose applied; U. lactuca displayed a higher number of biomarker candidates, including those in distinct metabolic pathways, increasing its usefulness for ecotoxicological applications. By evaluating the potential application of optical and biochemical traits, it was evident that the fatty acid profiles of the different exposure groups are excellent candidates in P. tricornutum, concomitant with the characteristics of this anionic surfactant. On the other hand, the results presented by laser-induced fluorescence and some parameters of PAM fluorometry in U. lactuca may be an advantage in the field, offering non-invasive, fast, easy-to-use, high-throughput screening techniques as excellent tools for ecotoxicology assessment.
- Editorial: Aquaculture of emergent marine invertebrates: Advances in nutrition, rearing technology and end-product qualityPublication . Lourenço, Sílvia; Valente, Luísa M. P.Polyphenols exhibit a wide range of biological and pharmacological activities, yet their dermal delivery is often limited by skin permeability and formulation-dependent constraints. In this work, the potential of roller microneedling as a physical strategy to improve dermal delivery of representative polyphenols—epicatechin, epigallocatechin gallate (EGCG), and curcumin—was investigated using common topical dosage forms, namely polymeric gel, patch, and ointment. Standardized skin permeation parameters were first obtained using excised porcine skin under conditions of stratum corneum saturation. The microneedling procedure was characterized, and its effect on polyphenol transdermal permeation from each formulation type was evaluated using Franz diffusion cells. Maximum steady-state permeation fluxes varied from 13.2 μg/cm2/h (curcumin) to 708 μg/cm2/h (epicatechin). Roller microneedling produced well-defined microchannels across the stratum corneum, with a mean interchannel distance of 2.3 mm, without compromising skin integrity, and significantly enhanced polyphenol permeation from all tested vehicles. Depending on the compound and formulation, microneedling increased permeation fluxes and/or reduced lag times, with particularly relevant effects observed for gel-delivered curcumin and ointment-delivered EGCG. Moreover, the polyphenols exhibited a robust antioxidant activity (equivalent to micromolar ascorbic acid) after transdermal permeation, including following microneedle-assisted delivery, supporting the compatibility of microneedling and the formulation vehicles with polyphenol functionality. Overall enhancement factors were consistent, yielding approximately twofold increases in both permeation flux and antioxidant activity. These findings demonstrate that roller microneedling is a practical, low-invasive approach to enhance the dermal delivery of polyphenols from conventional topical formulations, offering a versatile strategy that complements formulation-based optimization for therapeutic and cosmetic applications.
- Effect of stocking density and different diets on growth of Percula Clownfish, Amphiprion percula (Lacepede, 1802)Publication . Chambel, João; Severiano, Vera; Baptista, Teresa; Mendes, Susana; Pedrosa, RuiThe aim of this study was to evaluate the influence of stocking density (0.5, 1, 2 and 3 fishL−1) and commercial marine fish diets (diet A, B, C and D) over four months on specific growth rate, condition factor, percentage without anomalous pigmentation (partial or total lack of white bands -miss-band) and survival of juvenile Amphiprion percula. Results showed that at 0.5 fishL−1 densities induced the best survival (100%) and also the maximum percentage of fish without miss-band (58.33 +/−4.417%). The maximum SGR was obtained for the 0.5 fishL−1 (0.459 ± 0.023% cm/day). However, the best condition factor (2.53 +/− 0.27) was achieved for 2 fishL−1 densities. There were no significant differences in survival (68.9 to 84.5%), fish without miss-bands (18.03 to 26.92%) and condition factor (1.92 to 2.1) among diets during the experimental period. On the other hand, diet C (with 41% crude protein) supported the best SGR (0.485 ± 0.001% cmday−1). The results suggested that stocking density are critical and more relevant when compared with the different diet tested, namely on specific growth rate, condition factor, the miss-band and survival of juvenile percula clownfish. This study has particular significance with regards to anemonefishes husbandry in terms of survival and production efficiency.
- Effects of cadmium and resource quality on freshwater detritus processing chains: a microcosm approach with two insect speciesPublication . Campos, Diana; Alves, Artur; Lemos, Marco F. L.; Correia, António; Soares, Amadeu M. V. M.; Pestana, João L. T.Detritus processing is vital for freshwater ecosystems that depend on the leaf litter from riparian vegetation and is mediated by microorganisms and aquatic invertebrates. Shredder invertebrates transform coarse particulate organic matter into fine particulate organic matter used as food by collector species. Direct and indirect effects of contaminants can impair detritus processing and thus affect the functioning of these ecosystems. Here, we assessed the combined effects of a toxic metal (cadmium) and resource quality (leaf species) on detritus processing and shredder-collector interactions. We considered two types of leaves, alder and eucalyptus that were microbially conditioned under different Cd concentrations in the laboratory. The microbial communities present on leaves were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE), and we also measured microbial respiration rates. Sericostoma vittatum (a caddisfly shredder) and Chironomus riparius (a midge collector) were also exposed to Cd and allowed to consume the corresponding alder or eucalyptus leaves. We evaluated C. riparius growth and leaf mass loss in multispecies microcosms. Cadmium exposure affected leaf conditioning and fungal diversity on both leaf species, as assessed by DGGE. Cadmium exposure also affected the mass loss of alder leaves by reductions in detritivore feeding, and impaired C. riparius growth. Chironomus riparius consumed alder leaf discs in the absence of shredders, but S. vittatum appear to promote C. riparius growth in treatments containing eucalyptus. These results show that indirect effects of contaminants along detritus-processing chains can occur through effects on shredder-collector interactions such as facilitation but they also depend on the nutritional quality of detritus and on sensitivity and feeding plasticity of detritivore species.
- Evaluating the Potential of the Defatted By-Product of Aurantiochytrium sp. Industrial Cultivation as a Functional FoodPublication . Reboleira, João; Félix, Rafael; Félix, Carina; Melo, Marcelo M. R. de; Silva, Carlos M.; Saraiva, Jorge A.; Bandarra, Narcisa M.; Teixeira, Bárbara; Mendes, Rogério; Paulo, Maria C.; Coutinho, Joana; Lemos, Marco F. L.While Aurantiochytrium sp. is an increasingly popular source of polyunsaturated fatty acids (PUFAs), its extraction generates high amounts of waste, including the spent, defatted residue. The composition and bioactivities of this by-product could prove to be a major part of the sustainable valorisation of this organism within the framework of a circular economy. In this study, the defatted biomass of commercial Aurantiochytrium sp. was nutritionally characterised, and its amino acid profile was detailed. Additionally, the antioxidant and prebiotic potentials of an enzymatically digested sample of defatted Aurantiochytrium sp. were evaluated under a set of miniaturised in vitro assays. The nutritional profile of the spent Aurantiochytrium biomass revealed a protein and dietary-fibre rich product, with values reaching 26.7% and 31.0% for each, respectively. It also held high concentrations of glutamic and aspartic acid, as well as a favourable lysine/arginine ratio of 3.73. The digested samples demonstrated significant Weissela cibaria and Bifidobacterium bifidum growth-enhancing potential. Residual ferric reducing antioxidant power (FRAP) activity was likely attributed to antioxidant amino acids or peptides. The study demonstrated that some of the nutritional and functional potential that reside in the defatted Aurantiochytrium sp. waste encourages additional studies and the development of food supplements employing this resource’s by-products under a biorefinery framework.
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