Oligonucleotide cyclization: The thiol-maleimide reaction revisited

A novel method to synthesize cyclic oligonucleotides (5- to 26-mer) using the thiol-maleimide reaction is described. The target molecules were obtained after subsequent removal of thiol and maleimide protecting groups from 5′-maleimido-3′-thiol-derivatized linear precursors. Retro-Diels-Alder conditions deprotecting the maleimide simultaneously promoted cyclization cleanly and in high yield

El nuevo espacio Web del Atlas Nacional de España, antesala del SIANE

Este artículo presenta los resultados fruto del reciente convenio conjunto entre el Instituto Geográfico Nacional (IGN) y la Universidad Politécnica de Madrid (UPM), que ha tenido por objeto la investigación, diseño y creación de las páginas Web del Atlas Nacional de España (ANE). De carácter complementario a los objetos y datos que constituyen el ANE, este nuevo espacio Web que aquí se presenta constituirá el acceso al Sistema de Información del Atlas Nacional de España (SIANE), proporcionado la base tecnológica para la futura gestión y actualización del ANE. En definitiva, se trata de crear un marco contextual que por un lado haga comprender rápidamente al usuario qué puede ofrecerle SIANE, y por otro lado, complementar y potenciar la gestión informativa de éste presentando otros contenidos, diferentes a los objetos y datos publicados en el Atlas Nacional de España, que permitan definir y caracterizar esta importante obra

Short-term responses to fatty acids on lipid metabolism and adipogenesis in rainbow trout (Oncorhynchus mykiss)

Fish are rich in n-3 long-chain polyunsaturated fatty acids (LC-PUFA) such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Due to the increasing use of vegetable oils (VO), their proportion in diets has lowered, affecting lipid metabolism and fillet composition. Rainbow trout cultured preadipocytes were treated with representative FA found in fish oils (EPA and DHA) or VO (linoleic, LA and alpha-linolenic, ALA acids), while EPA and LA were also orally administered, to evaluate their effects on adipogenesis and lipid metabolism. In vitro, all FA increased lipid internalization, with ALA producing the highest effect, together with upregulating the FA transporter fatp1. In vivo, EPA or LA increased peroxisome proliferator-activated receptors ppara and pparb transcripts abundance in adipose tissue, suggesting elevated β-oxidation, contrary to the results obtained in liver. Furthermore, the increased expression of FA synthase (fas) and the FA translocase/cluster of differentiation (cd36) in adipose tissue indicated an enhanced uptake of lipids and lipogenesis de novo, whereas stable or low hepatic expression of genes involved in lipid transport and turnover was found. Thus, fish showed a similar tissue metabolic response to the short-term availability of EPA or LA in vivo, while in vitro VO-derived FA demonstrated greater potential inducing fat accumulation

Musculoskeletal growth modulation in gilthead sea bream juveniles reared at high water temperature and fed with palm and rapeseed oils-based diets

The upward trend of seawater temperature has encouraged improving the knowledge of its consequences on fish, considering also the development of diets including vegetable ingredients as an approach to achieve a more sustainable aquaculture. This study aims to determine the effects on musculoskeletal growth of: (1) a high-water temperature of 28 °C (versus 21 °C) in gilthead sea bream juveniles (Sparus aurata) fed with a diet rich in palm oil and, (2) feeding the fish reared at 28 °C with two other diets containing rapeseed oil or an equilibrated combination of both vegetable oils. Somatic parameters and mRNA levels of growth hormone-insulin-like growth factors (GH-IGFs) axis-, osteogenic-, myogenic-, lipid metabolism- and oxidative stress-related genes in vertebra bone and/or white muscle are analyzed. Overall, the data indicate that high-water rearing temperature in this species leads to different adjustments through modulating the gene expression of members of the GH-IGFs axis (down-regulating igf-1, its receptors, and binding proteins) and also, to bone turnover (reducing the resorption-activity genes cathepsin K (ctsk) and matrix metalloproteinase-9 (mmp9)) to achieve harmonic musculoskeletal growth. Moreover, the combination of palm and rapeseed oils seems to be the most beneficial at high-water rearing temperature for both balanced somatic growth and muscular fatty acid uptake and oxidation

Genomic and Phenotypic Agreement Defines the Use of Microwave Dielectric Spectroscopy for Recording Muscle Lipid Content in European Seabass (Dicentrarchus labrax)

Recording the fillet lipid percentage in European seabass is crucial to control lipid deposition as a means toward improving production efficiency and product quality. The reference method for recording lipid content is solvent lipid extraction and is the most accurate and precise method available. However, it is costly, requires sacrificing the fish and grinding the fillet sample which limits the scope of applications, for example grading of fillets, recording live fish or selective breeding of fish with own phenotypes are all limited. We tested a rapid, cost effective and non-destructive handheld microwave dielectric spectrometer (namely the Distell fat meter) against the reference method by recording both methods on 313 European seabass (Dicentrarchus labrax). The total method agreement between the dielectric spectrometer and the reference method was assessed by Lin's concordance correlation coefficient (CCC), which was low to moderate CCC = 0.36-0.63. We detected a significant underestimation in accuracy of lipid percentage 22-26% by the dielectric spectrometer and increased imprecision resulting in the coefficient of variation (CV) doubling for dielectric spectrometer CV = 40.7-46% as compared to the reference method 27-31%. Substantial genetic variation for fillet lipid percentage was found for both the reference method (h 2 = 0.59) and dielectric spectroscopy (h 2 = 0.38-0.58), demonstrating that selective breeding is a promising method for controlling fillet lipid content. Importantly, the genetic correlation (rg) between the dielectric spectrometer and the reference method was positive and close to unity (rg = 0.96), demonstrating the dielectric spectrometer captures practically all the genetic variation in the reference method. These findings form the basis of defining the scope of applications and experimental design for using dielectric spectroscopy for recording fillet lipid content in European seabass and validate its use for selective breedin

Diet and exercise modulate gh-igfs axis, proteolytic markers and myogenic regulatory factors in juveniles of gilthead sea bream (Sparus aurata)

© 2021 by the authors.The physiological and endocrine benefits of sustained exercise in fish were largely demonstrated, and this work examines how the swimming activity can modify the effects of two diets (high-protein, HP: 54% proteins, 15% lipids; high-energy, HE: 50% proteins, 20% lipids) on different growth performance markers in gilthead sea bream juveniles. After 6 weeks of experimentation, fish under voluntary swimming and fed with HP showed significantly higher circulating growth hormone (GH) levels and plasma GH/insulin-like growth-1 (IGF-1) ratio than fish fed with HE, but under exercise, differences disappeared. The transcriptional profile of the GH-IGFs axis molecules and myogenic regulatory factors in liver and muscle was barely affected by diet and swimming conditions. Under voluntary swimming, fish fed with HE showed significantly increased mRNA levels of capn1, capn2, capn3, capns1a, n3, and ub, decreased gene and protein expression of Ctsl and Mafbx and lower muscle texture than fish fed with HP. When fish were exposed to sustained exercise, diet-induced differences in proteases’ expression and muscle texture almost disappeared. Overall, these results suggest that exercise might be a useful tool to minimize nutrient imbalances and that proteolytic genes could be good markers of the culture conditions and dietary treatments in fish.This study was supported by the projects from the “Ministerio de Economía y Competitividad” (MINECO) AGL2015-70679-R and RTI2018-100757-B-I00 to J.G. and J.B., and the “Xarxa de Refèrencia d’R+D+I en Aqüicultura” and the 2017SGR1574 from the “Generalitat de Catalunya”. M.P.- A., I.G.-P. and E.J.V. were supported by predoctoral fellowships from the MINECO, BES-2016-078697, PRE2019-089578 and BES-2013-062949, respectively

Proteolytic systems' expression during myogenesis and transcriptional regulation by amino acids in gilthead sea bream cultured muscle cells

Proteolytic systems exert an important role in vertebrate muscle controlling protein turnover, recycling of amino acids (AA) or its use for energy production, as well as other functions like myogenesis. In fish, proteolytic systems are crucial for the relatively high muscle somatic index they possess, and because protein is the most important dietary component. Thus in this study, the molecular profile of proteolytic markers (calpains, cathepsins and ubiquitin-proteasome system (UbP) members) were analyzed during gilthead sea bream (Sparus aurata) myogenesis in vitro and under different AA treatments. The gene expression of calpains (capn1, capn3 and capns1b) decreased progressively during myogenesis together with the proteasome member n3; whereas capn2, capns1a, capns1b and ubiquitin (ub) remained stable. Contrarily, the cathepsin D (ctsd) paralogs and E3 ubiquitin ligases mafbx and murf1, showed a significant peak in gene expression at day 8 of culture that slightly decreased afterwards. Moreover, the protein expression analyzed for selected molecules presented in general the same profile of the mRNA levels, which was confirmed by correlation analysis. These data suggest that calpains seem to be more important during proliferation, while cathepsins and the UbP system appear to be required for myogenic differentiation. Concerning the transcriptional regulation by AA, the recovery of their levels after a short starvation period did not show effects on cathepsins expression, whereas it down-regulated the expression of capn3, capns1b, mafbx, murf1 and up-regulated n3. With regards to AA deficiencies, the major changes occurred at day 2, when leucine limitation suppressed ctsb and ctsl expression. Besides at the same time, both leucine and lysine deficiencies increased the expression of mafbx and murf1 and decreased that of n3. Overall, the opposite nutritional regulation observed, especially for the UbP members, points out an efficient and complementary role of these factors that could be useful in gilthead sea bream diets optimization

Effects of different dietary vegetable oils on growth and intestinal performance, lipid metabolism and flesh quality in gilthead sea bream

This study tests the effects of feeding different vegetable oils (VO) in gilthead sea bream with the aim of improving sustainable aquafeeds. Juveniles were fed ad libitum with ten isonitrogenous (46%) and isolipidic (22%) diets with a 75% fish oil (FO) replacement, differing in VO composition leading to two experimental blocks: fish fed with VO blends or mono-substituted diets. Growth parameters, skin and muscle colour, muscle texture, plasma metabolites, digestive capacities, and transcript levels of intestinal lipid transport, muscle dynamics and lipid metabolism-related genes in white muscle and adipose tissue were studied. In fish fed high-palm oil diets, final body weight and mesenteric fat significantly increased, while effects were not found in hepatosomatic index, reflecting tissue-specific lipid accumulation. Relative intestinal length increased significantly with dietary soya oil (SO) content, suggesting a compensatory mechanism to improve nutrient absorption capacity. Plasma parameters showed few changes upon dietary treatments. Lipase activity was unaltered, while intestinal fatp1b expression increased in animals fed blended diets high in rapeseed oil (RO). In adipose tissue, expression of nuclear receptors pparβ and lxr was modified by dietary fatty acids (FA) profile; however, regarding lipid metabolism and β-oxidation genes, only lpl showed significant differences, suggesting that FA uptake and oxidation, but not de novo lipogenesis is what appears to determine the increase in adipose tissue mass. In fish fed blended VO diets, lpl expression showed a positive correlation with MUFA dietary content, suggesting that some FA present in RO enhance its expression, according to data from fish fed mono-substituted diets. In muscle, fish fed blended VO diets also showed a positive correlation of lpl expression with dietary MUFA, whereas in mono-substituted, it was significantly higher in fish fed SO, suggesting other mechanisms are involved in LPL regulation. Concerning β-oxidation genes in muscle, significant differences were detected in cpt1a expression for fish fed blended VO diets, whereas hadh and cpt1b were unaltered, suggesting slight FA uptake regulation in mitochondria. Expression levels of genes related to myogenic processes were not greatly modified by dietary lipid sources except for myogenin in blended VO diets-fed fish, showing a similar profile as that in body weight and opposite with the differentiation marker mhc. This study provides new information regarding the effects of dietary VO, demonstrating moderate effects in lipid homeostasis without adverse effects on growth performance, leading to a transversal view of the responses and interactions from intestine to muscle growth and flesh quality

Mitochondrial Adaptation to Diet and Swimming Activity in Gilthead Seabream: Improved Nutritional Efficiency

Sustained exercise promotes growth in different fish species, and in gilthead seabream we have demonstrated that it improves nutrient use efficiency. This study assesses for differences in growth rate, tissue composition and energy metabolism in gilthead seabream juveniles fed two diets: high-protein (HP; 54% protein, 15% lipid) or high energy (HE; 50% protein, 20% lipid), under voluntary swimming (VS) or moderate-to-low-intensity sustained swimming (SS) for 6 weeks. HE fed fish under VS conditions showed lower body weight and higher muscle lipid content than HP fed fish, but no differences between the two groups were observed under SS conditions. Irrespective of the swimming regime, the white muscle stable isotopes profile of the HE group revealed increased nitrogen and carbon turnovers. Nitrogen fractionation increased in the HP fed fish under SS, indicating enhanced dietary protein oxidation. Hepatic gene expression markers of energy metabolism and mitochondrial biogenesis showed clear differences between the two diets under VS: a significant shift in the COX/CS ratio, modifications in UCPs, and downregulation of PGC1a in the HE-fed fish. Swimming induced mitochondrial remodeling through upregulation of fusion and fission markers, and removing almost all the differences observed under VS. In the HE-fed fish, white skeletal muscle benefited from the increased energy demand, amending the oxidative uncoupling produced under the VS condition by an excess of lipids and the pro-fission state observed in mitochondria. Contrarily, red muscle revealed more tolerant to the energy content of the HE diet, even under VS conditions, with higher expression of oxidative enzymes (COX and CS) without any sign of mitochondrial stress or mitochondrial biogenesis induction. Furthermore, this tissue had enough plasticity to shift its metabolism under higher energy demand (SS), again equalizing the differences observed between diets under VS condition. Globally, the balance between dietary nutrients affects mitochondrial regulation due to their use as energy fuels, but exercise corrects imbalances allowing practical diets with lower protein and higher lipid content without detrimental effects

Regulatory mechanisms involved in muscle and bone remodeling during refeeding in gilthead sea bream

The tolerance of fish to fasting offers a model to study the regulatory mechanisms and changes produced when feeding is restored. Gilthead sea bream juveniles were exposed to a 21-days fasting period followed by 2h to 7-days refeeding. Fasting provoked a decrease in body weight, somatic indexes, and muscle gene expression of members of the Gh/Igf system, signaling molecules (akt, tor and downstream effectors), proliferation marker pcna, myogenic regulatory factors, myostatin, and proteolytic molecules such as cathepsins or calpains, while most ubiquitin-proteasome system members increased or remained stable. In bone, downregulated expression of Gh/Igf members and osteogenic factors was observed, whereas expression of the osteoclastic marker ctsk was increased. Refeeding recovered the expression of Gh/Igf system, myogenic and osteogenic factors in a sequence similar to that of development. Akt and Tor phosphorylation raised at 2 and 5h post-refeeding, much faster than its gene expression increased, which occurred at day 7. The expression in bone and muscle of the inhibitor myostatin (mstn2) showed an inverse profile suggesting an inter-organ coordination that needs to be further explored in fish. Overall, this study provides new information on the molecules involved in the musculoskeletal system remodeling during the early stages of refeeding in fish