CD19 LYMPHOCYTE PROLIFERATION INDUCED BY Bifidobacterium animalis subsp. lactis IN C57BL/6 MICE EXPERIMENTALLY INFECTED WITH Toxoplasma gondii

Toxoplasmosis is frequently acquired through the oral route by the ingestion of cysts or oocysts of Toxoplasma gondii. Once ingested, the parasites penetrate the intestinal epithelial cells and rapidly disseminate to all organs in the host. During T. gondii infection, the intestinal microbiota plays an important role in stimulating a protective immune response against the parasite. In this sense the use of probiotics is worthy of note since they are live microorganisms that have beneficial effects on the host through stimulation of the immune response that can be important in the control of T. gondii proliferation and dissemination in the host. In the present study, the action of the probiotic Bifidobacterium animalis subsp. lactis was investigated in C57BL/6 mice infected with oocysts of ME49 strain of T. gondii. The probiotic had an immunomodulatory action, inducing CD19 lymphocyte proliferation and consequently increasing anti-T. gondii antibody level.Bifidobacterium animalis subsp. lactisprovided protection in supplemented mice, compared to the control group. In addition, supplemented animals had milder inflammatory process in the small intestine, indicating that the probiotic protects the intestinal mucosa during infection with T. gondii. It was concluded that the probioticB. animalis subsp. lactis induces humoral immune response capable of providing protection against T. gondii infection

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N(1)-glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N(1)-(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.info:eu-repo/semantics/publishedVersio

Contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.info:eu-repo/semantics/publishedVersio

Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.info:eu-repo/semantics/publishedVersio

Prescrição de Benzodiazepinas e outros Sedativos na Administração Regional de Saúde de Lisboa e Vale do Tejo de 2013 a 2020: Um Estudo Retrospetivo

Introdução: Portugal é o país da Organização para a Cooperação e Desenvolvimento Económico com maior consumo de ansiolíticos, hipnóticos e sedativos, sendo uma proporção significativa constituída por benzodiazepinas ou análogos, associados a efeitos de tolerância e dependência. Por este motivo, em alternativa às benzodiazepinas para tratamento da insónia, algumas publicações identificam outros fármacos com efeito hipnótico, como antidepressivos, anti-histamínicos, antipsicóticos ou anticonvulsivantes. Assim, torna-se necessário compreender a evolução do consumo destes medicamentos, pelo que foi objetivo deste estudo avaliar a evolução da dispensa de benzodiazepinas, outros fármacos ansiolíticos, hipnóticos ou sedativos não benzodiazepínicos, fármacos com potencial uso off-label na insónia e os resultados de indicadores dos Cuidados de Saúde Primários neste âmbito na região de Lisboa e Vale do Tejo. Material e Métodos: Realizou-se um estudo em base de dados, censitário e retrospetivo, no período de 2013 até 2020, avaliando-se a evolução das variáveis total de doses diárias definidas, doses diárias definidas por 1000 habitantes por dia (DHD) e dos indicadores relevantes. Os dados foram extraídos da plataforma SIARS da Administração Regional de Saúde de Lisboa e Vale do Tejo. Resultados: Verificou-se uma diminuição da dispensa de benzodiazepinas (de 57,44 para 51,77 DHD) mas o aumento da dispensa de não benzodiazepinas e de fármacos com potencial uso off-label (de 6,56 para 8,56 DHD e de 14,70 para 25,92 DHD, respetivamente). O zolpidem foi o mais dispensado entre os fármacos não benzodiazepínicos, acompanhando a tendência crescente de dispensa (de 4,86 para 6,96 DHD). Do conjunto de fármacos com potencial para uso off-label verificaram-se aumentos da dispensa para a trazodona (de 3,81 para 7,92 DHD), mirtazapina (de 3,52 para 6,48 DHD), pregabalina (de 3,15 para 4,87 DHD), quetiapina (de 2,68 para 4,59 DHD) e gabapentina (de 1,32 para 1,90 DHD), mas mais significativo ou apenas verificado nas formulações com dosagem mais baixa. A mediana dos resultados do indicador “proporção de idosos sem prescrição de sedativos, ansiolíticos e hipnóticos” em 2015 foi de 81,0, tendo em 2020 aumentado para 84,9. A mediana do indicador “proporção de utentes sem prescrição prolongada de ansiolíticos, sedativos e hipnóticos” em 2019 foi de 93,6 e aumentou para 94,3 em 2020. Conclusão: Globalmente, verificou-se uma redução da dispensa de benzodiazepinas prescritas na Região de Lisboa e Vale do Tejo. Parece existir uma alteração do padrão de prescrição no tratamento da insónia. São necessários estudos mais robustos para confirmar esta observação

The characterization of Escherichia coli CpdB as a recombinantpProtein reveals that, besides having the expected 3´-nucleotidase and 2´,3´-cyclic mononucleotide phosphodiesterase activities, it is also active as cyclic dinucleotide phosphodiesterase

Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´- cyclicmononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´- cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the ‘average’ enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.Trabajo financiado por: Junta de Extremadura y Fondos FEDER. Ayudas GR10133 y GR15143 Donación privada para María Jesús Costas Vázquez. Contrato 2015/00481/001peerReviewe

O Paleolítico de Melgaço: Vestígios arqueológicos dos primeiros habitantes do Concelho

info:eu-repo/semantics/publishedVersio

How do students perceive their Learning Assessment?

The present work was developed considering the learning assessment vector and linking it to students’ general satisfaction with the engineering course. The key findings show a positive association between students’ general satisfaction and the learning assessment, specifically with the assessment methodologies effectiveness regarding the different subjects taught. Globally,studentsconsidered the assessment methodologies effective and appropriate to the different subjects taught however, they do not feel encouraged to be part of the process of monitoring his/her performance, and to thetime allocated to the assessment testsas it wasnot considered appropriateto the volume of subjects taught.The authors would like to express their acknowledgments to all students who accepted to collaborate in this study. The authors also thank the Research Centre CIETI and FCT, for all the support provided in the scope of the projects COMPETE: POCI-01-0145-FEDER-007043, UID/CEC/00319-2019 and UID-EQU-04730-2013.N/

Ocupações pleistocénicas da margem esquerda do Baixo Minho (Miño/Minho2): objetivos e primeiros resultados de um projeto transfronteiriço

As mais antigas investigações arqueológicas no âmbito do Paleolítico da bacia hidrográfica do rio Minho (NW Peninsular) iniciaram-se na primeira metade do século XX. Por esta mesma altura realizaram-se também os primeiros estudos geomorfológicos na região, que permitiram correlacionar artefactos líticos com terraços fluviais.Durante a segunda metade daquele mesmo século, o estudo do Paleolítico ocorreu essencialmente na Galiza, ficando em Portugal reduzido a trabalhos pontuais e geograficamente circunscritos. A partir de 2010 assiste-se, igualmente na Galiza, ao desenvolvimento de um projecto de investigação que possibilitou não só a identificação de novos sítios arqueológicos, como também a sua datação absoluta, remetendo-os para o Plistocénico médio. A elaboração do projecto Miño-Minho, cujos primeiros resultados agora se apresentam, teve como principal objectivo dar continuidade na margem portuguesa a estes trabalhos iniciados no país vizinho.The earliest archaeological research on the Palaeolithic of the Minho River Basin (NW Iberia) took place in the first half of the 20th century. At the same time, geomorphological studies were developed in the region allowing the connection between lithic artefacts and fluvial terraces. In the second half of this century the studies on the Palaeolithic occurred mainly in Galicia, while in Portugal they were short-term and focused on geographically confined areas. From 2010 onwards, also in Galicia, a research project was developed which enabled the detection of new archaeological sites and their absolute dating, assigning them to the Middle Pleistocene. Thedevelopment of the Miño-Minho project, whose first results are now presented, had as its main objective to carry on in the Portuguese bank the research initiated in the neighboring country