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- Análise de notificações RASFF e sua perspetiva evolutiva no quadriénio 2020-2023 no contexto portuguêsPublication . Cameira, Margarida Mendes da Fonseca Santos; Rodrigues, Cristina Maria Baptista; Ferreira, Marília Catarina Leal FazeresPara garantir um elevado padrão no que se refere à segurança dos alimentos, é necessário identificar os perigos a que os géneros alimentícios, materiais para contacto com alimentos e alimentos para animais estão sujeitos, efetuar uma avaliação de risco e desenvolver um conjunto de medidas reativas e proativas de acordo com as conclusões retiradas. O RASFF é uma ferramenta que auxilia neste processo, ao possibilitar a troca rápida de informações para que as autoridades ajam em conformidade e impeçam que produtos não seguros cheguem aos consumidores. Este estudo incide sobre as notificações RASFF em que Portugal esteve envolvido entre 2020 e 2023 e pretende-se inferir a sua evolução e qual o contexto português, assinalando os pontos fortes e os que careçam de especial atenção. Desta forma, realizou-se uma pesquisa na base de dados RASFF Window e analisaram-se os dados obtidos consoante o tipo da notificação, seguida das categorias de perigo. Apurou-se um predomínio das notificações sobre géneros alimentícios (577/656, 88% nos distribuídos, 178/208, 85,6% nos notificados e 76/80, 95% nos com origem em Portugal), relativamente aos materiais para contacto com os alimentos e os alimentos para animais. Como categorias de perigo mais importantes nos géneros alimentícios distribuídos para Portugal salientam-se os “resíduos de pesticidas” (201/587, 34,2%) e os “microrganismos patogénicos” (77/587, 13,1%), tendo a última apresentado uma maior relevância no ano final do quadriénio estudado em detrimento da “resíduos de pesticidas”. Quanto aos géneros alimentícios com origem em Portugal, “metais pesados” foi a categoria de perigo mais contabilizada (14/78, 18%), seguindo-se “biotoxinas (outras)” e “resíduos de pesticidas” (11/78, 14% cada uma). Da totalidade dos produtos que deram entrada no espaço europeu através dos postos de controlo fronteiriços localizados no território nacional e que foram considerados não conformes e notificados no RASFF, a grande maioria, 92,9% (117/126), foi detetada durante o controlo de fronteiras e os restantes, posteriormente em controlos de rotina quando já se encontravam no mercado. No que respeita aos produtos nacionais afetados, a percentagem de notificações emitidas pelas autoridades portuguesas (10/80, 13%), enquadra-se no funcionamento do sistema europeu, em que todos os Estados-Membros partilham responsabilidades na deteção e comunicação de não conformidades
- Analysis of conservation priorities of Iberoamerican cattle based on autosomal microsatellite markersPublication . Ginja, Catarina; Gama, Luís T.; Cortes, Óscar; Delgado, Juan Vicente; Dunner, Susana; García, David; Landi, Vincenzo; Martín-Burriel, Inmaculada; Martínez-Martínez, Amparo; Penedo, M. Cecília T.; Rodellar, Clementina; Zaragoza, Pilar; Cañon, Javier; BioBovis ConsortiumBackground: Determining the value of livestock breeds is essential to define conservation priorities, manage genetic diversity and allocate funds. Within- and between-breed genetic diversity need to be assessed to preserve the highest intra-specific variability. Information on genetic diversity and risk status is still lacking for many Creole cattle breeds from the Americas, despite their distinct evolutionary trajectories and adaptation to extreme environmental conditions. Methods: A comprehensive genetic analysis of 67 Iberoamerican cattle breeds was carried out with 19 FAOrecommended microsatellites to assess conservation priorities. Contributions to global diversity were investigated using alternative methods, with different weights given to the within- and between-breed components of genetic diversity. Information on Iberoamerican plus 15 worldwide cattle breeds was used to investigate the contribution of geographical breed groups to global genetic diversity. Results: Overall, Creole cattle breeds showed a high level of genetic diversity with the highest level found in breeds admixed with zebu cattle, which were clearly differentiated from all other breeds. Within-breed kinships revealed seven highly inbred Creole breeds for which measures are needed to avoid further genetic erosion. However, if contribution to heterozygosity was the only criterion considered, some of these breeds had the lowest priority for conservation decisions. The Weitzman approach prioritized highly differentiated breeds, such as Guabalá, Romosinuano, Cr. Patagonico, Siboney and Caracú, while kinship-based methods prioritized mainly zebu-related breeds. With the combined approaches, breed ranking depended on the weights given to the within- and between-breed components of diversity. Overall, the Creole groups of breeds were generally assigned a higher priority for conservation than the European groups of breeds. Conclusions: Conservation priorities differed significantly according to the weight given to within- and betweenbreed genetic diversity. Thus, when establishing conservation programs, it is necessary to also take into account other features. Creole cattle and local isolated breeds retain a high level of genetic diversity. The development of sustainable breeding and crossbreeding programs for Creole breeds, and the added value resulting from their products should be taken into consideration to ensure their long-term survival.
- Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for Gluco- and Galacto- Substituted mannans and Its calcium induced stabilityPublication . Ghosh, Arabinda; Luís, Ana Sofia; Brás, Joana L. A.; Pathaw, Neeta; Chrungoo, Nikhil K.; Fontes, Carlos M. G. A.; Goyal, ArunThis study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.
- Thermostable Recombinant β‑(1→4)-Mannanase from C. thermocellum: biochemical characterization and manno-oligosaccharides productionPublication . Ghosh, Arabinda; Luís, Ana Sofia; Brás, Joana L. A.; Fontes, Carlos M. G. A.; Goyal, ArunFunctional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum ATCC 27405. Its sequence comparison the exhibited highest similarity with Man26B of C. thermocellum F1. The full length CtManf and truncated CtManT were cloned in the pET28a(+) vector and expressed in E. coli BL21(DE3) cells, exhibiting 53 kDa and 38 kDa proteins, respectively. On the basis of the substrate specificity and hydrolyzed product profile, CtManf and CtManT were classified as β-(1→4)-mannanase. A 1.5 fold higher activity of both enzymes was observed by Ca2+ and Mg2+ salts. Plausible mannanase activity of CtManf was revealed by the classical hydrolysis pattern of carob galactomannan and the release of manno-oligosaccharides. Notably highest protein concentrations of CtManf and CtManT were achieved in tryptone yeast extract (TY) medium, as compared with other defined media. Both CtManf and CtManT displayed stability at 60 and 50 °C, respectively, and Ca2+ ions imparted higher thermostability, resisting their melting up to 100 °C.
- Heterosis for meat quality and fatty acid profiles in crosses among Bos indicus and Bos taurus finished on pasture or grainPublication . Gama, L.T.; Bressan, M.C.; Rodrigues, E.C.; Rossato, L.V.; Moreira, O.C.; Alves, S.P.; Bessa, R.J.B.Physicochemical properties and fatty acid profiles of meat from Bos indicus, Bos taurus and crossbred B. taurus× B. indicus bullocks (n=216), finished on pasture or grain, were used to estimate the effects of heterosis. Meat quality and fatty acid profiles generally benefitedwith crossbreeding, but the advantages from heterosis differed among finishing systems. The Warner–Bratzler shear-force in fresh and aged meat was reduced due to heterosis in pasture-finishing, but the effect was minor under grain-finishing. With pasture-finishing, heterosis caused an increase of 5% in CLA concentration, but few other changes in fatty acid profiles. In grain-finishing, heterosis caused a reduction in intramuscular fat and cholesterol, increased amounts of PUFA, n−6 fatty acids and PUFA/SFA ratio, and a decline in atherogenic index. The Δ9 desaturase estimated activity in crossbreds showed a behavior close to B. indicus, suggesting the existence of few loci and a dominance genetic effect on enzymes involved in fatty acid synthesis and metabolism.
- Genetic structure, relationships and admixture with wild relatives in native pig breeds from Iberia and its islandsPublication . Gama, Luis T.; Martínez, Amparo M.; Carolino, Inês; Landi, Vincenzo; Delgado, Juan V.; Vicente, Antonio A.; Vega-Pla, José L.; Cortés, Oscar; Sousa, Conceição O.; BIOPIG ConsortiumBackground: Native pig breeds in the Iberian Peninsula are broadly classified as belonging to either the Celtic or the Mediterranean breed groups, but there are other local populations that do not fit into any of these groups. Most of the native pig breeds in Iberia are in danger of extinction, and the assessment of their genetic diversity and population structure, relationships and possible admixture between breeds, and the appraisal of conservation alternatives are crucial to adopt appropriate management strategies. Methods: A panel of 24 microsatellite markers was used to genotype 844 animals representing the 17 most important native swine breeds and wild populations existing in Portugal and Spain and various statistical tools were applied to analyze the results. Results: Genetic diversity was high in the breeds studied, with an overall mean of 13.6 alleles per locus and an average expected heterozygosity of 0.80. Signs of genetic bottlenecks were observed in breeds with a small census size, and population substructure was present in some of the breeds with larger census sizes. Variability among breeds accounted for about 20% of the total genetic diversity, and was explained mostly by differences among the Celtic, Mediterranean and Basque breed groups, rather than by differences between domestic and wild pigs. Breeds clustered closely according to group, and proximity was detected between wild pigs and the Mediterranean cluster of breeds. Most breeds had their own structure and identity, with very little evidence of admixture, except for the Retinto and Entrepelado varieties of the Mediterranean group, which are very similar. Genetic influence of the identified breed clusters extends beyond the specific geographical areas across borders throughout the Iberian Peninsula, with a very sharp transition from one breed group to another. Analysis of conservation priorities confirms that the ranking of a breed for conservation depends on the emphasis placed on its contribution to the betweenand within-breed components of genetic diversity. Conclusions: Native pig breeds in Iberia reveal high levels of genetic diversity, a solid breed structure and a clear organization in well-defined clusters.
- Multiple rewards from a treasure trove of novel glycoside hydrolase and polysaccharide lyase structures : new folds, mechanistic details, and evolutionary relationshipsPublication . Fushinobu, Shinya; Alves, Victor D.; Coutinho, Pedro MRecent progress in three-dimensional structure analyses of glycoside hydrolases (GHs) and polysaccharide lyases (PLs), the historically relevant enzyme classes involved in the cleavage of glycosidic bonds of carbohydrates and glycoconjugates, is reviewed. To date, about 80% and 95% of the GH and PL families, respectively, have a representative crystal structure. New structures have been determined for enzymes acting on plant cell wall polysaccharides, sphingolipids, blood group antigens, milk oligosaccharides, Nglycans, oral biofilms and dietary seaweeds. Some GH enzymes have very unique catalytic residues such as the Asp- His dyad. New methods such as high-speed atomic force microscopy and computational simulation have opened up a path to investigate both the dynamics and the detailed molecular interactions displayed by these enzymes.
- Genetic diversity, antibiotic resistance and biofilm-forming ability of Arcobacter butzleri isolated from poultry and environment from a Portuguese slaughterhousePublication . Ferreira, Susana; Fraqueza, Maria J.; Queiroz, João A.; Domingues, Fernanda C.; Oleastro, MónicaThe genus Arcobacter is an emerging pathogen associated with several clinical symptoms. This genus is widely distributed and has been isolated from environmental, animal, food and human samples, where poultry is considered the major source. In this study, forty three Arcobacter butzleri strains isolated from poultry and environment of a Portuguese slaughterhouse, were characterized by pulsed field gel electrophoresis (PFGE) and assessed for antimicrobial susceptibility and ability to form biofilms. PFGE patterns obtained using restriction enzymes SmaI and SacII revealed high genetic diversity, with 32 distinct PFGE patterns. Most of A. butzleri isolates presented multiple antimicrobial resistance, exhibiting four different resistance profiles. All 43 isolates were susceptible to gentamicin and 2.3% were resistant to chloramphenicol, in contrast to twenty four (55.8%) that were resistant to ciprofloxacin. Among 36 selected isolates, 26 strains presented biofilm-forming ability, which was dependent on the atmosphere and initial inoculum density. Overall, the results showed that A. butzleri displays a high genetic diversity, and presents resistance to several antibiotics, which together with its biofilm formation ability may represent a potential hazard for foodborne infections and a considerable risk for human health.
- Small angle X-ray scattering analysis of Clostridium thermocellum cellulosome N-terminal complexes reveals a highly dynamic structurePublication . Currie, Mark A.; Cameron, Kate; Dias, Fernando M. V.; Spencer, Holly L.; Bayer, Edward A.; Fontes, Carlos M. G. A.; Smith, Steven P.; Jia, ZongchaoClostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.
- Biocide and antimicrobial susceptibility of methicillin-resistant staphylococcal isolates from horsesPublication . Couto, Natacha; Belas, Adriana; Tilley, Paula; Couto, Isabel; Gama, Luís T.; Kadlec, Kristina; Schwarz, Stefan; Pomba, ConstançaThe aim of this study was to evaluate the biocide and antimicrobial susceptibility of methicillin-resistant staphylococcal isolates from horses. Fourteen methicillin-resistant staphylococci (MRS) were subjected to an extensive genotype characterization, including SCCmec, dru, spa, PFGE and MLST typing. Antimicrobial susceptibility testing was performed and resistance genes were detected by PCR. Minimum bactericidal concentrations (MBCs) of four biocides [chlorhexidine acetate (CHA), benzalkonium chloride (BAC), triclosan (TCL) and glutaraldehyde (GLA)] were determined following the recommendations of document NF EN 1040. The presence of qac and sh-fabI genes was investigated by PCR. Several antimicrobial resistance patterns and genes were detected. When MRS strains were exposed for a longer period of time, a lower concentration of the biocide was needed to achieve lethality. TCL had the lowest MBC values. All MBC values were lower than the recommended in-use concentrations for veterinary medicine. S. haemolyticus and S. cohnii subsp. cohnii carried plasmid-borne qacA and sh-fabI or qacB and a qacH-like genes, respectively. Biocides appear to be a reliable antiseptic option against MRS, since even in the presence of bacterial efflux mechanisms, the recommended concentration is much higher than the in vitro MBC.
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