Orthophosphate uptake in proteoid roots of naturally occurring Hakea sericea Schrad.

Poster apresentado no congresso "14th Congress of the Federation of European Societies of Plant Biology", August 23-27, 2004, Cracow, Poland.P is an important macronutrient, making up about 0.2% of a plant’s dry weight. The form of P most readily accessed by plants is Pi, the concentration of which rarely exceeds 10 µM in soil (Schachtman et al., 1998). Here we studied the mechanisms involved on extraction of Pi from soil in Hakea sericea Schrad. (Proteacea), an Australian invader of natural habitats, which is able to develop proteoid roots as a response to P deficiency. Material and Methods: Proteoid roots were harvested from adult H. sericea shrubs growing in Serra d’Arga, Northern Portugal. The P uptake buffer contained 2.5-4000 µM NaH2PO4 and Pi concentration was determined according to Adams (1991). PCR amplification was performed using genomic DNA and degenerate primers based on the most conserved regions of plant P transporters genes. Results and Conclusions: H. sericea proteoid roots have highly efficient transporters for extraction of Pi from soil. Kinetic studies supported the involvement of two Pi mediated transport systems associated with a diffusion-like mechanism. The kinetic parameters were as follows: for the high-affinity system Km, 11 µM Pi and Vmax, 7 µmol h-1 g-1 FW; for the low-affinity system Km, 163 µM Pi and Vmax, 30 µmol h-1 g-1 FW. The protonophore CCCP (50 µM) inhibited up to 60% the initial uptake rates of 5-50 µM Pi (high-affinity range) and 100-500 µM (low-affinity range), suggesting H+-dependent transport. Both transporters were competitively inhibited by phosphite and 100 µM mersalyl reduced by 50% the initial uptake rates of 10 µM Pi. PCR using degenerate primers allowed the amplification of two 438 bp fragments sharing 87.4% identity with each other. Both nucleotide sequences comprise an incomplete ORF coding for transmembrane domains and exhibiting 85-82% identity with other plant P transporters. These nucleotide sequences are being used for the identification of complete cDNAs of Pi transporter genes of H. sericea.Fundação para a Ciência e a Tecnologia (FCT) - Bolsa Ref. SFRH/BD/10899/2002 , Bolsa Ref. SFRH/BD/13460/2003

Hakea sericea Schrad. - a model to study phosphate uptake in proteoid roots

Comunicação em painel apresentada no XIV Congresso Nacional de Bioquímica, 2 a 4 Dezembro 2004, Vilamoura.P is an important macronutrient, making up about 0.2% of a plant dry weight. Pi is the form of P most readily accessed by plants, the concentration of which rarely exceeds 10 µM in soil. Here we studied the mechanisms involved on Pi uptake from soil in Hakea sericea Schrad. (Proteacea), an Australian invader of natural habitats, able to develop proteoid roots as a response to P deficiency. H. sericea proteoid roots have efficient transporters for extraction of Pi from soil. Uptake studies supported the involvement of two Pi transport systems with the following kinetic parameters: for the high-affinity system Km, 6 µM Pi and Vmax, 5 µmolh-1g-1 FW; for the low-affinity system Km, 100 µM Pi and Vmax, 24 µmolh-1g-1 FW. The protonophore CCCP (50 µM) inhibited up to 60% the initial uptake rates of 5-50 µM Pi (high-affinity range) and 100-500 µM (low-affinity range), suggesting H+-dependent transport. Both transporters were competitively inhibited by phosphite and 100 µM mersalyl reduced by 50% the initial uptake rates of 10 µM Pi. PCR using degenerate primers designed for the conserved regions of Pi transporter genes from higher plants allowed the amplification of two 437 bp fragments sharing 77,4% identity with each other. Both nucleotide sequences comprise an incomplete ORF coding for transmembrane domains and exhibit homology with other plant Pi transporters. These nucleotide sequences are being used as probes in the identification of complete sequences of Pi transporter genes in H. sericea genome.Fundação para a Ciência e a Tecnologia (FCT) - grant ref. SFRH/BD/10899/2002 , grant ref. SFRH/BD/13460/2003

Human phenylalanine hydroxylase as the case study

Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. The authors would like to thank Luís Miguel Ramos and Cátia Nascimento who contributed to the exploratory research that culminated in the work herein presented. Funding Information: Authors acknowledge Sofarimex, Indústria Química e Farmacêutica SA, Portugal, for all the support concerning freeze-drying studies. This work was supported by FEDER and Fundação para a Ciência e a Tecnologia, I. P. through iMED.ULisboa (Projects UIDB/04138/2020 and UIDP/04138/2020), iNOVA4Health (UIDB/04462/2020, UIDP/04462/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020) and research project PTDC/EBB-BIO/101237/2008 and research grant SFRH/BD/47946/2008 (to Paulo R. Lino). This work has also received funding from the National PKU Alliance, USA. Publisher Copyright: © 2023 The Author(s)The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.publishersversionpublishe

Neuroinfection survey at a neurological ward in a Brazilian tertiary teaching hospital

OBJECTIVES: This study was undertaken to characterize the neuroinfection profile in a tertiary neurological ward. INTRODUCTION: Neuroinfection is a worldwide concern and bacterial meningitis, tetanus and cerebral malaria have been reported as the commonest causes in developing countries. METHODS: From 1999 to 2007, all patients admitted to the Neurology Ward of Hospital das Clínicas, São Paulo University School of Medicine because of neuroinfection had their medical records reviewed. Age, gender, immunological status, neurological syndrome at presentation, infectious agent and clinical outcome were recorded. RESULTS: Three hundred and seventy four cases of neuroinfectious diseases accounted for 4.2% of ward admissions and the identification of infectious agent was successful in 81% of cases. Mean age was 40.5 + 13.4 years, 63.8% were male, 19.7% were immunocompromised patients and meningoencephalitis was the most common clinical presentation despite infectious agent. Viruses and bacteria were equally responsible for 29.4% of neuroinfectious diseases; parasitic, fungal and prion infections accounted for 28%, 9.6% and 3.5% respectively. Human immunodeficiency virus (HIV), herpes simplex virus 1 (HSV1), Mycobacterium tuberculosis, Treponema pallidum, Taenia solium, Schistosoma mansoni, Cryptococcus neoformans and Histoplasma capsulatum were the more common infectious pathogens in the patients. Infection mortality rate was 14.2%, of which 62.3% occurred in immunocompetent patients. CONCLUSION: Our institution appeared to share some results with developed and developing countries. Comparison with literature may be considered as quality control to health assistance

Acerca de uma leitura geopolítica das relações entre Portugal e o Atlântico

O artigo pretende traçar uma breve perspectiva geopolítica da relação entre Portugal e o oceano Atlântico. Essa relação, embora sempre presente e importante, variou ao longo da nossa histó- ria. O autor, depois de tentar sumariamente caracterizar essas variações, analisa simplificadamente aquela relação no actual contexto do sistema global das relações internacionais. Dessa análise retira argumentos para afirmar que na ligação com o Atlântico poderão de novo ser encontradas as soluções mais adequadas para os nossos actuais problemas do desenvolvimento e da afirmação internacional, através da possível e desejável futura assumpção por Portugal de um papel de entreposto de importantes fluxos nas relações transatlânticas, em que as luso-brasileiras e as com a CPLP deverão desempenhar um papel centra

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis

Funding Information: This work was supported by European Regional Development Fund (FEDER) through the Operational Program for Competitiveness Factors (COMPETE) [HealthyAging2020 CENTRO-01-0145-FEDER-000012-N2323, POCI-01-0145-FEDER-016385, POCI-01-0145-FEDER-007440 to CNC.IBILI, POCI-01-0145-FEDER-007274 to i3S/INEB and NORTE-01-0145-FEDER-000012 to T.L.L.]; national funds through the Portuguese Foundation for Science and Technology (FCT) [PTDC/SAU-ORG/119296/2010, PTDC/ NEU-OSD/0312/2012, PESTC/ SAU/UI3282/2013-2014, MITP-TB/ECE/0013/ 2013, FCT-UID/NEU/04539/2013], PD/BD/52294/2013 to T.M.R.R., SFRH/ BD/85556/2012 (co-financed by QREN) to V.C.S]; Lisboa Portugal Regional Operational Programme (LISBOA 2020) and Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement; and by INFARMED Autoridade Nacional do Medicamento e Produtos de Saúde, I.P. [FIS-FIS-2015-01_CCV_20150630-157]. Publisher Copyright: © 2017 The Author.Aims Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.publishersversionpublishe

Alterações morfométricas na retina, coroide e nervo ótico após infeção por SARS-CoV-2

O novo coronavírus responsável pela síndrome respiratória aguda grave (SARS-CoV- 2) surgiu associado à pandemia por COVID-19. A enzima conversora da angiotensina 2 (ACE-2), com aparente importância na COVID-19, pela interação com as proteínas na superfície do vírus, tem expressão em vários tecidos oculares e várias alterações como conjuntivite, uveíte, vasculite e neurite foram descritas inicialmente em modelos animais. Em estudos mais recentes, embora na maioria em doentes COVID-19 moderados/grave, tem sido descrito o comprometimento da superfície ocular anterior e do polo posterior reforçando a ideia de neurotropismo (pela facilidade de envolvimento do sistema nervoso central) que classicamente é descrito em outros coronavirus. Algumas alterações do polo posterior incluem o compromisso vascular/ isquémico tornando relevante também a observação da coroide. A SARS-CoV-2 tem sido associada à diminuição das camadas internas da retina e à presença de lesões hiperrefletivas, micro-hemorragias e manchas algodonosas. No entanto, o envolvimento da retina e a coróide em doentes previamente infetados com COVID-19 ainda não é totalmente compreendido. De forma a clarificar o envolvimento dos fatores de neuro-degeneração e vasculares, descritos em indivíduos recuperados de COVID-19 moderada/grave, é fundamental perceber que alterações existem ao nível da retina interna e da coroide, em indivíduos recuperados de COVID-19 ligeira. Questão de investigação: Existem alterações morfométricas da retina, coroide e nervo ótivo em indivíduos recuperados de COVID-19 ligeira?info:eu-repo/semantics/publishedVersio

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.Peer Reviewe