Pelo olhar de quem não sente a diferença : comparação dos Cores Sets da Classificação Internacional de Funcionalidade, Incapacidade e Saúde (CIF) para a Paralisia Cerebral com a perspetiva de crianças/ jovens e cuidadores, de Portugal

Introdução: A CIF foi considerada, por alguns clínicos, como não sendo totalmente viável para a aplicação na rotina clínica, já que este sistema de classificação contém mais de 1400 categorias. Posto isto, o processo de desenvolvimento de Core Sets da CIF tem sido priorizado pela OMS, tornando a CIF acessível para a avaliação das cs específicas. Estes pretendem orientar uma avaliação multidisciplinar abrangente, incluindo as categorias da CIF para descrever, de uma forma suficiente, a funcionalidade e incapacidade dos indivíduos. Contudo, podem existir divergências entre a perspetiva de crianças e jovens portugueses com PC e cuidadores sobre a sua cs e os core sets da CIF estabelecidos para a mesma. Objetivo: Identificar os problemas percecionados pelas crianças e jovens com paralisia cerebral e pelos cuidadores em Portugal e analisar em que medida estes aspetos são representados pela versão atual do Comprehensive Core Set da CIF para esta cs. Metodologia: Este estudo é qualitativo, tendo-se utilizado uma amostra conveniente de crianças/jovens com PC e cuidadores. Todos os dados foram recolhidos por meio de entrevistas e posteriormente analisados segundo o protocolo de Coenen. Foi efetuado o linking dos conceitos obtidos das entrevistas para as categorias da CIF, tendo sido comparadas com os Core Sets da CIF para esta condição. Resultados: Este estudo tem uma amostra de 48 indivíduos, sendo que 28 eram crianças e jovens com PC e os restantes 20 eram cuidadores. A média de idades das crianças/jovens incluídos foi 13,04 (±3,26), sendo que a idade mínima incluída foi 8 anos e a idade máxima, 18 anos. A média de idades dos cuidadores incluídos foi de 35,20 (±8,01) anos, sendo que a idade mínima incluída foi 22 anos e a idade máxima 55 anos. Das 256 categorias identificadas, 101 estão contempladas nas categorias dos Comprehensive Core Set da CIF. Estas encontram-se presentes em 58 categorias de 2º nível dos Comprehensive Core Set de um total de 135, correspondendo a 43% dos mesmos. Conclusões: Tendo em conta os resultados obtidos, podemos concluir que os Comprehensive Core Set da CIF para a PC contemplam a grande maioria dos problemas percecionados pela população-alvo, ainda que integre várias categorias que não foram relevantes para esta população em estudo

The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome

Funding Information: We thank Prof. Carlos Fontes and Dr Joana Bras (NZYTech, Portugal) for their assistance in obtaining the initial BT0996-C clone. We are grateful to Prof Ten Feizi, Dr Yan Liu and Dr Lisete Silva from the Glycosciences Laboratory (Imperial College London, UK) for their support and assistance on robotic microarray printing. This work was supported by the FCT - Fundação para a Ciência e a Tecnologia, I.P., through the DL-57/2016 Program Contract (BP). This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. The authors acknowledge the European Synchrotron Radiation Facility (Grenoble, France) and ALBA (Barcelona, Spain) for access to beamlines ID30B and BL-13 XALOC, respectively. Publisher Copyright: © 2022The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.publishersversionpublishe

The crystal structure of the R280K mutant of human p53 explains the loss of DNA binding

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.We thank Gilberto Fronza (from Mutagenesi e Prevenzione Oncologica, Ospedale Policlinico San Martino, Genova, Italy), for providing us with the pLS76 vector. We acknowledge the European Synchrotron Radiation Facility for the provision of synchrotron radiation facilities and access to beamline ID30B. This work received financial support from the European Union (FEDER, Fundo Europeu de Desenvolvimento Regional, funds POCI/01/0145/FEDER/007728 through Programa Operacional Factores de Competitividade–COMPETE) and National Funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) under the Partnership Agreement PT2020 UID/MULTI/04378/2013, and projects (3599-PPCDT) PTDC/DTP-FTO/1981/2014–POCI-01-0145-FEDER-016581 and RECI/BBB-BEP/0124/2012. FCT fellowships: PD/BD/114046/2015 (Ana Sara Gomes) and SFRH/BD/96189/2013 (Sara Gomes) (thanks FCT PhD Doctoral Programme BiotechHealth), and SFRH/BPD/110640/2015 (Carla Oliveira).info:eu-repo/semantics/publishedVersio

Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus

This work was supported by Fundação para a Ciência e a Tecnologia (FCT-MCTES), Portugal, through project grant PTDC/BIA-MIB/31730/2017 (to A.S.P.), fellowships PD/BD/105727/2014 (to V.G.C.) and SFRH/BD/143494/2019 (to F.T.), and program contract DL-57/2016 (to B.A.P. and C.N.) and by Wellcome Trust Biomedical Resource grants number WT108430/Z/15/Z and WT218304/Z/19/Z, a March of Dimes (Arlington, VA, USA) Prematurity Research Center grant (number 22-FY18-821) for the funding to the Carbohydrate Microarray Facility, Associate Laboratory projects LAQV-REQUIMTE (UIDB/50006/2020) and CICECO-Aveiro Institute of Materials (UIDB/50011/2020 & UIDP/50011/2020), and by the Applied Molecular Biosciences Unit (UCIBIO), which is financed by Portuguese national funds from FCT-MCTES (UIDP/04378/2020 and UIDB/04378/2020).A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.publishersversionpublishe

A purification platform for antibodies and derived fragments using a de novo designed affinity adsorbent

Fundação para a Ciência e Tecnologia (FCT) -PTDC/BII-BIO/28878/2017; co-financed by ERDF under the PT2020 Partnership Agreement LISBOA-01-0145-FEDER-028878; PD/BD/128251/2016 from MIT-PT PhD Bioengineering Systems to MJBM; SFRH/BPD/112543/2015 to AJMB; Applied Molecular Biosciences Unit – UCIBIO (UIDP/04378/2020 ; UIDB/04378/2020); CICECO-Aveiro Institute of Materials (UIDB/50011/2020; UIDP/50011/2020), financed by national funds from FCT.Antibody technologies are the most representative success-case in the biopharmaceutical industry. Widely available purification technologies fail in providing a dedicated universal purification platform that can accommodate antibodies structural diversity, namely antibodies from non-human sources, as chicken IgY, and antigen-binding fragments. In this work, we took inspiration from natural and engineered antibody-binding ligands, to rationally design affinity adsorbents able to capture full-length antibodies and fragments. The one-pot Petasis and Ugi combinatorial reactions were sequentially employed to rapidly generate a library of putative solid-phase adsorbents. The best performing adsorbent yielded a single-step recovery, under mild conditions, of human and chicken whole antibodies, antigen-binding fragments and engineered single-domain antibodies from different complex feedstocks. Due to its simple preparation, the lead antibody adsorbent finds broad applicability as a universal purification platform to increase the availability of antibody technologies in research and development.authorsversionpublishe