Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling—An In Vitro Study

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes’ immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1ß and nf-¿b expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-¿b and p65. This study showed the ability of methionine surplus to improve leucocytes’ response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.This work was partially supported by UIDB/04423/2020, UIDP/ 04423/2020 and INFLAMMAA (reference PTDC/CVT-CVT/ 32349/2017), financed by Portugal and the European Union through FEDER and COMPETE 2020, and national funds through Fundação para a Ciência e a Tecnologia (FCT, Portugal). MM and BC were supported by FCT, Portugal (SFRH/BD/108243/2015 and IF/00197/2015, respectively)

Rearranjos Subteloméricos: Apresentação de 21 Probandos, com Ênfase nos Casos Familiares

Intellectual disability affects 2% - 3% of the general population, with a chromosomal abnormality being found in 4% - 28% of these patients and a cryptic subtelomeric abnormality in 3% - 16%. In most cases, these subtelomeric rearrangements are submicroscopic, requiring techniques other than conventional karyotype for detection. They may be de novo or inherited from an affected parent or from a healthy carrier of a balanced chromosomal abnormality. The aim of this study was to characterize patients from our medical genetics center, in whom both a deletion and duplication in subtelomeric regions were found.Introdução:O défice intelectual afeta 2% – 3% da população geral, sendo encontrada uma alteração cromossómica em 4% – 28% dos casos e uma alteração subtelomérica em 3% – 16%. Estas alterações subteloméricas são, na maioria dos casos, submicroscópicas, não sendo detetadas no cariótipo convencional. Podem ser de novoou herdadas de um progenitor afetado ou de um progenitor saudável portador de um rearranjo equilibrado. O objetivo deste estudo foi caracterizar os doentes seguidos no nosso centro de genética médica com uma deleção e uma duplicação nas regiões subteloméricas. Material e Métodos:Caracterização clínica e citogenética de 21 probandos com alterações subteloméricas seguidos no nosso centro entre 1998 e 2017. Resultados:Foram caracterizados 21 probandos que apresentavam défice intelectual e dismorfia facial, pertencentes a 19 famílias. Sete tinham alterações do comportamento, cinco epilepsia e 14 outro sinal ou sintoma. Quatro tinham alterações no cariótipo e quatro foram diagnosticados por array-comparative genomic hybridization. Em quatro famílias não foi possível o estudo dos progenitores. Quando um dos fenótipos era dominante (síndrome de deleção ou duplicação), foi atribuída a classificação online mendelian inheritance in man. Discussão:Foi realizada classificação dos doentes e das famílias. As alterações nas regiões subteloméricas são, apesar de raras, uma causa substancial para défice intelectual sindrómico com repercussões familiares importantes. É essencial lembrar que um arraycomparative genomic hybridizationnormal não exclui um rearranjo equilibrado familiar. Conclusão:O estudo dos progenitores é essencial não só para caracterização completa do rearranjo mas também para um aconselhamento genético preciso e identificação de familiares em risco de recorrência. Palavras-chave:Deficiência Intelectual/genética; Rearranjo Génico/genética; Telómero/genéticainfo:eu-repo/semantics/publishedVersio

Extraction of antioxidant compounds from enzymatic hydrolysis of brewer's spent grain after solid-state fermentation

Phenolic compounds have antioxidant properties that can benefit human health and food preservation. During solid-state fermentation (SSF) of lignocellulosic substrates, filamentous fungi produce carbohydrate degrading enzymes which can amplify the quantity of phenolics released linked to vegetable matrix when applying enzymatic hydrolysis (EH). In this work, brewer's spent grain (BSG) was fermented with Aspergillus ibericus and later the lignocellulolytic enzymes were extracted and used on an EH of fermented BSG. EH was optimized by a Box-Behnken design approach, studying the effect of three factors in the extraction of antioxidant compounds, namely the quantity of SSF-crude extract added, load of fermented solid and addition of -glucosidase. The maximum increase of phenols and antioxidant activity in comparison to unhydrolyzed BSG were 42.9 mg/g and 83.5 mol trolox/g, respectively. -glucosidase addition and low solid-load had a positive effect on the phenols released and antioxidant activity. The quantities of crude SSF-extract studied did not had a significant effect on phenols and antioxidant released.Helena Fernandes was supported by PhD grant (SFRH/BD/131219/2017). Funded by the European Regional Development Fund (ERDF) under the Project SPO3 (ref.: POCI-01-0145-FEDER030377) BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the ERDF under the scope of Norte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Solid-state fermentation of winery and olive mill wastes for lignocellulolytic enzymes and antioxidant compounds production

Solid-state fermentation (SSF) is a process that can use agroin-dustry solid residues as substrate for microbial growth. During SSF, value-added products and fermented biomass are generated con-tributing for the reutilization of otherwise pollutant agroindustry wastes. By SSF, fungi can hydrolyze anti-nutritional factors of plant-based residues, as the lignocellulosic matrix, and produce enzymes, phenolic compounds and other bioactive compounds, making this a high interest bioprocess for the aquafeed industry. The present study optimized (simplex centroid design) the mixture of winery and olive mill wastes to produce lignocellulolytic enzymes and to extract phenolic compounds through SSF by Aspergillus niger. The substrate mixture that maximized the production of lignocellu-lolytic enzymes, antioxidant and phenolic compounds was 0.44 g exhausted olive pomace/gds; 0.3 g exhausted grape marc/gds; 0.15 g vine-shoot trimmings and 0.1 g crude olive pomace/gds. The crude extract will be applied as additive in meagre (Argyrosomus regius) diets, to improve the feed utilization efciency of low-sh meal-based diets, contributing to the sustainability of aquafeeds.Funded by Programa Operacional Mar2020, Portugal 2020 under the project InovFeed (ref.MAR-02.01.01-FEAMP-0111) and BioTec Norte operation (NORTE-01-0145-FEDER-000004) funded by ERDF under the scope of Norte 2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Chromatographic techniques for the determination of free phenol in foundry resins

Phenol is a toxic compound present in a wide variety of foundry resins. Its quantification is important for the characterization of the resins as well as for the evaluation of free contaminants present in foundry wastes. Two chromatographic methods, liquid chromatography with ultraviolet detection (LC-UV) and gas chromatography with flame ionization detection (GC-FID), for the analysis of free phenol in several foundry resins, after a simple extraction procedure (30 min), were developed. Both chromatographic methods were suitable for the determination of phenol in the studied furanic and phenolic resins, showing good selectivity, accuracy (recovery 99–100%; relative deviations <5%), and precision (coefficients of variation <6%). The used ASTM reference method was only found to be useful in the analysis of phenolic resins, while the LC and GC methods were applicable for all the studied resins. The developed methods reduce the time of analysis from 3.5 hours to about 30 min and can readily be used in routine quality control laboratories

Effect of short chain fructooligosaccharides (scFOS) on immunological status and gut microbiota of gilthead sea bream (Sparus aurata) reared at two temperatures

The effects of dietary short chain fructooligosaccharides (scFOS) incorporation on hematology, fish immune status, gut microbiota composition, digestive enzymes activities, and gut morphology, was evaluated in gilthead sea bream (Sparus aurata) juveniles reared at 18 °C and 25 °C. For that purpose, fish with 32 g were fed diets including 0, 0.1, 0.25 and 0.5% scFOS during 8 weeks. Overall, scFOS had only minor effects on gilthead sea bream immune status. Lymphocytes decreased in fish fed the 0.1% scFOS diet. Fish fed the 0.5% scFOS diet presented increased nitric oxide (NO) production, while total immunoglobulins (Ig) dropped in those fish, but only in the ones reared at 25 °C. Red blood cells, hemoglobin, bactericidal activity and NO were higher at 25 °C, whereas total white blood cells, circulating thrombocytes, monocytes and neutrophils were higher at 18 °C. In fish fed scFOS, lymphocytes were higher at 18 °C. Total Ig were also higher at 18 °C but only in fish fed 0.1% and 0.5% scFOS diets. No differences in gut bacterial profiles were detected by PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) between dietary treatments. However, group's similarity was higher at 25 °C. Digestive enzymes activities were higher at 25 °C but were unaffected by prebiotics incorporation. Gut morphology was also unaffected by dietary prebiotic incorporation. Overall, gut microbiota composition, digestive enzymes activities and immunity parameters were affected by rearing temperature whereas dietary scFOS incorporation had only minor effects on these parameters. In conclusion, at the tested levels scFOS does not seem worthy of including it in gilthead sea bream juveniles diets.This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT Foundation for Science and Technology, under the projects "PEst-C/MAR/LA0015/2013 and UID/Multi/04423/2013". I. Guerreiro, P. Enes, A. Couto and B. Costas were supported by grants (SFRH/BD/76139/2011; BPD/39688/2007, BD/47495/2008 and BPD/77210/2011, respectively) from FCT, Portugal. C. Serra and A. Salvador were recipient of grants within the Project AQUAIMPROV (reference NORTE-07-0124-FEDER-000038) and Project BioEnv (REF. NORTE-07-0124-FEDER-000048), co-financed by the North Portugal Regional Operational Programme (ON.2 - O Novo Norte). We would like to express our thanks to Pedro Correia for the assistance during the growth trial. Authors would also thank to Jefo Species-specific additives France, for providing the prebiotic

Identification of a mosaic non-inherited small supernumerary ring chromosome 2: cytogenetic-molecular studies and genotype-phenotype correlation

Introduction: The identification of supernumerary marker chromosomes (SMCs) derived from all the autosomes is currently possible, but rarely by conventional cytogenetics alone. Supernumerary ring chromosomes (SRCs) account for about 10% of these cases. SRCs derived from chromosome 2 are unusual, and there are only a few cases reported in the literature. The severity of the phenotype depends on the type of the mosaicism, the percentage of cells affected by the genetic change and the chromosome involved. Methods: The authors report the case of a boy aged 8 referred for cytogenetic studies, presenting with behavior and learning problems, mental retardation with uncoordenated speech, attention deficit and hyperactivity (PHDA), as well as small slanting palpebral fissures. The karyotype was obtained from peripheral blood lymphocyte cultures using high resolution GTL banding and standard techniques. Fluorescence in situ hybridization (FISH) was performed using specific probes for the centromeric regions of all chromosomes (Chromoprobe Multiprobe - ISystem). Results: Cytogenetic analysis revealed two cell lines: one with a supernumerary marker ring chromosome, 47,XY,+r (52%), and a normal cell line, 46,XY (48%). The SRC was identified by FISH with the chromosome 2 centromeric probe. Since the parents had normal karyotypes, this abnormality was “de novo”. Final karyotype of the proband was: mos 47,XY,+r[26]/46,XY[24].ish r(2)(D2Z2+)dn. Discussion: The clinical description of this patient is in agreement with other reports of the literature. Molecular characterization by FISH analyses is an useful way of investigating the presence of euchromatin contained in a SMC and establishing new chromosomal syndromes. However, to better characterize this ring, in order to establish a more accurate genotype-phenotype correlation, more studies involving other technologies should be performed, thus allowing suitable genetic counsellin

Bioaccessibility and intestinal uptake of minerals from different types of home-cooked and ready-to-eat beans

Limited information exists on the bioaccessibility and intestinal uptake of essential minerals from different types of beans cooked through different cooking methods. This study aimed to estimate the in vitro bioaccessibility and intestinal uptake of the essential minerals K, Ca, Mg, Fe, Zn, Mn, Cu and Mo from four different types of beans (kidney, black, white and cowpea) cooked under different conditions (home-cooked and industrially processed canned product). Results showed that the bioaccessibility and uptake of most essential minerals is higher in canned beans. Mn was the mineral that presented the highest bioaccessibility (43–63%), and Mo had the lowest (3–36%). The highest uptake was observed for Mg (59.9%), while the lowest was found for Fe (10.5%). Regarding the type of beans, white beans presented the highest bioaccessibility and uptake for all the analyzed essential minerals and thus its consumption is preferable from a nutritional point of view.Edgar Pinto and César Oliveira acknowledge Instituto de Ciências e Tecnologias Agrárias e Agro-Alimentares da Universidade do Porto (ICETA) for their contracts NORTE-01-0145-FEDER-0001 and NORTE-01-0145-FEDER-000011, respectively. This work received financial support from the European Union (POCI/01/0145/FEDER/007265), from the European Regional Development Fund (ERDF) through COMPETE – Operational Competitiveness Programme and from national funds provided by FCT – Foundation for Science and Technology under the project UID/QUI/50006/2013.info:eu-repo/semantics/publishedVersio