18,089 research outputs found

    Mechanisms of As, Cd, Pb, and Zn hyperaccumulation by plants and their effects on soil microbiome in the rhizosphere

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    Excess potentially toxic elements (PTEs), including arsenic (As), cadmium (Cd), lead (Pb), and zinc (Zn), above permissible limits in the environment, have detrimental effects on trophic levels. Hence, imperative to devise advertent measures to address this situation, especially in the soil ecosystem: the major reservoir of many PTEs. Using aerial plant parts (shoot) to accumulate As, Cd, Pb, and Zn - hyperaccumulators are considered a permanent approach to PTE removal from soils. This communication expatiated the principles that govern the hyperaccumulation of plants growing on As, Cd, Pb, and Zn-contaminated soils. The contribution of soil microbial communities during hyperaccumulation is well-elaborated to support the preference for this remediation approach. The study confirms a flow direction involving PTE uptake‚Äďtranslocation‚Äďtolerance‚Äďdetoxification by hyperaccumulators. Rhizosphere microbes exhibit a direct preference for specific hyperaccumulators, which is associated with root exudations, while the resultant formation of chelates and solubility of PTEs, with soil physicochemical properties, including pH and redox potential, promote uptake. Different compartments of plants possess specialized transporter proteins and gene expressions capable of influx and efflux of PTEs by hyperaccumulators. After PTE uptake, many hyperaccumulators undergo cellular secretion of chelates supported by enzymatic catalysis and high transport systems with the ability to form complexes as tolerance and detoxification mechanisms. The benefits of combining hyperaccumulators with beneficial microbes such as endophytes and other rhizosphere microbes for PTE removal from soils are vital in enhancing plant survival and growth, minimizing metal toxicity, and supplying nutrients. Inoculation of suitable rhizosphere microbes can promote efficient cleaning of PTEs contaminated sites utilizing hyperaccumulator plants

    Urinary biomarkers of biofortified beef in healthy women explored by untargeted metabolomics

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    Background: The prevalence of overweight and non-communicable chronic diseases is rising all over the globe. The high consumption of energy dense foods on behalf of high nutrient-dense food leads to lower intake of essential vitamins and minerals, such as vitamins D, E, K, and selenium. These micronutrients are related with numerous human vital functions and their deficiency is positively associated with higher risk of chronic diseases and mortality. Bovine meat is an important source of several micronutrients, with higher bioavailability compared to other plant-based foods. Meat consumption is expected to increase worldwide, therefore the biofortification of bull‚Äôs feeds can be an innovative strategy to increase population‚Äôs exposure to nutrients. Metabolomics techniques are capable to explore if the supplementation will ultimately lead to a higher micronutrient‚Äôs uptake in the body. Objective: The aim of the present study was to explore the differences on urinary metabolic fingerprint of women ingesting 300g of beef a day from bulls fed concentrate supplemented with extra vitamin D, E, K, and selenium compared to the regular composite feed. Methodology: A 32 days double-blind randomized cross-over human intervention study with two intervention periods, each for 6 days, was conducted in 35 healthy women. The participants were instructed to eat 300g of grinded beef meat as raw weight per day, either from bulls fed with regular control feed or meat supplemented with vitamin D, E, K and selenium, combined with their habitual diet. Fasting urine samples were collected in the morning before and after each intervention period and were analyzed by LC-MS untargeted metabolomics. Multivariate and univariate analysis were applied do identify discriminative features between the two interventions. Results: A total of 7 and 6 metabolites for positive and negative mode, respectively, were selected as discriminative of the two interventions. Among these, markers of overall meat intake, as well as markers of animal feed, markers related with the participants diet and inflammation-related markers were identified as upregulated or downregulated for the supplemented intervention. No markers specifically related to the biofortification were observed. Conclusions: Based on our methodology, the ingestion of biofortified beef did not results in a higher level of related metabolites when comparing the two interventions. Minor changes indicate that consequences of biofortification were very small. Further research is needed to understand if a higher increase of vitamin D, E, K, and selenium on animal¬īs feed composite can lead to different outcomes

    3D bioprinting of mineralizing cyanobacteria as novel approach for the fabrication of living building materials

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    Living building materials (LBM) are gaining interest in the field of sustainable alternative construction materials to reduce the significant impact of the construction industry on global CO2 emissions. This study investigated the process of three-dimensional bioprinting to create LBM incorporating the cyanobacterium Synechococcus sp. strain PCC 7002, which is capable of producing calcium carbonate (CaCO3) as a biocement. Rheology and printability of biomaterial inks based on alginate-methylcellulose hydrogels containing up to 50 wt% sea sand were examined. PCC 7002 was incorporated into the bioinks and cell viability and growth was characterized by fluorescence microscopy and chlorophyll extraction after the printing process. Biomineralization was induced in liquid culture and in the bioprinted LBM and observed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and through mechanical characterization. Cell viability in the bioprinted scaffolds was confirmed over 14 days of cultivation, demonstrating that the cells were able to withstand shear stress and pressure during the extrusion process and remain viable in the immobilized state. CaCO3 mineralization of PCC 7002 was observed in both liquid culture and bioprinted LBM. In comparison to cell-free scaffolds, LBM containing live cyanobacteria had a higher compressive strength. Therefore, bioprinted LBM containing photosynthetically active, mineralizing microorganisms could be proved to be beneficial for designing environmentally friendly construction materials

    Ferroptosis in hematological malignant tumors

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    Ferroptosis is a kind of iron-dependent programmed cell death discovered in recent years. Its main feature is the accumulation of lipid reactive oxygen species in cells, eventually leading to oxidative stress and cell death. It plays a pivotal role in normal physical conditions and the occurrence and development of various diseases. Studies have shown that tumor cells of the blood system, such as leukemia cells and lymphoma cells, are sensitive to the response to ferroptosis. Regulators that modulate the Ferroptosis pathway can accelerate or inhibit tumor disease progression. This article reviews the mechanism of ferroptosis and its research status in hematological malignancies. Understanding the mechanisms of ferroptosis could provide practical guidance for treating and preventing these dreaded diseases

    Identification of core cuprotosis-correlated biomarkers in abdominal aortic aneurysm immune microenvironment based on bioinformatics

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    BackgroundThe occurrence of abdominal aortic aneurysms (AAAs) is related to the disorder of immune microenvironment. Cuprotosis was reported to influence the immune microenvironment. The objective of this study is to identify cuprotosis-related genes involved in the pathogenesis and progression of AAA.MethodsDifferentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) in mouse were identified following AAA through high-throughput RNA sequencing. The enrichment analyses of pathway were selected through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). The validation of cuprotosis-related genes was conducted through immunofluorescence and western blot analyses.ResultsTotally, 27616 lncRNAs and 2189 mRNAs were observed to be differentially expressed (|Fold Change| ‚Č• 2 and q< 0.05) after AAA, including 10424 up-regulated and 17192 down-regulated lncRNAs, 1904 up-regulated and 285 down-regulated mRNAs. Gene ontology and KEGG pathway analysis showed that the DElncRNAs and DEmRNAs were implicated in many different biological processes and pathways. Furthermore, Cuprotosis-related genes (NLRP3, FDX1) were upregulated in the AAA samples compared with the normal one.ConclusionCuprotosis-related genes (NLRP3,FDX1) involved in AAA immune environment might be critical for providing new insight into identification of potential targets for AAA therapy

    Estudo da interação entre os filmes com base em quitosana e os componentes dos alimentos

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    Chitosan cross-linked with genipin (Ch-Ge) films are effective on white wine preservation, showing also positive aroma notes without changing wine typicity. The preservation process is explained by the decrease of the content of iron (50%) and copper (16%), inhibiting the oxidation reactions and microorganisms‚Äô growth. Wine positive aroma notes were related with the higher content of benzaldehyde and furfural, compounds that can be derived from Maillard reaction, evidencing that Ch-Ge films can participate in this reaction. Thus, the main objective of this PhD thesis was to understand the impact of these films on the preservation and composition of wine. The potential of Ch-Ge films in the formation of volatile products from Maillard reaction, as furfural, phenylacetaldehyde, and benzaldehyde, was studied using wine solutions. The results showed that films promoted the formation of furfural by dehydration of arabinose and the formation of phenylacetaldehyde and benzaldehyde, Strecker aldehydes, by phenylalanine degradation. The participation of Ch-Ge films in these reactions was explained by the amino groups of chitosan, which are involved in the formation of furfural from pentoses, whereas aldehyde groups react with amino acids to form Strecker aldehydes. As the content of benzaldehyde was much higher than the content of aldehydes derived from the reducing terminal of chitosan, it was shown that aldehydes groups from cross-linked genipin can also react with amino group of phenylalanine, thus promoting benzaldehyde formation. Model solutions of 2-amino-2-deoxy-D-glucitol, mimicking the structural unit of chitosan backbone, not presenting reducing sugar activity, were prepared, allowing to observe by tandem mass spectrometry with electrospray ionization the formation of an amide linkage grafting chitosan with genipin. To investigate whether Ch-Ge films can retain volatile compounds, wine model solutions were prepared containing the films and individual compounds representative of five chemical families, namely alcohols (hexanol), aldehydes (benzaldehyde and hexanal), carboxylic acids (hexanoic acid), esters (ethyl acetate and ethyl hexanoate), and ketones (octan-3-one). It was possible to verify that the retention capacity of the volatile compounds by Ch-Ge films increased with the hydrophobicity of compounds carbon chain. Beyond the hydrophobic interactions, ionic and other interactions were also observed, since carboxylic acids (54%, hexanoic acid) and esters (38%, ethyl hexanoate), followed by ketones (28%, octan-3-one) were also retained. To explain the Ch-Ge films capacity to retain iron ions, wine model solutions were prepared containing iron ions in the presence of Ch-Ge films in solutions acidified using tartaric acid or hydrochloric acid. The results demonstrated that Ch-Ge films only exhibited iron sorption capacity in the solutions containing tartaric acid. This observation triggered the study of other carboxylic acids, namely acetic, glycolic, lactic, succinic, malic, and citric acids. This study revealed that őĪ-hydroxypolycarboxylic acids, such as malic and citric acids promoted the ternary complexes formation in acidic media due to electrostatic attraction mechanism between protonated amino groups of chitosan and the negatively charged carboxylic acids of the iron-őĪ-hydroxypolycarboxylate complexes. The elucidation of the mechanism of iron sorption by Ch-Ge films triggered the modification of chitosan with carboxyl functional groups using diacetyl-L tartaric anhydride and galacturonic acid, allowing to create the N-tartarylCh-Ge and Ch-GalA-Ge films, respectively. The iron sorption capacity of these films was evaluated in acidic solutions without the presence of őĪ hydroxypolycarboxylic acids. However, the results revealed that both films did not interact with iron ions, possibly explained by the occurrence of intra and intermolecular electrostatic interactions between the positively charged chitosan amino groups and the negatively charged substituent groups, leading to the absence of free carboxylic groups in the Ch-Ge modified films. The remaining protonated amino groups seems to have promoted the repulsion of iron cations. Both chemical modifications of films lead to a decrease in the content of amino groups compared to Ch-Ge, allowing to expect that the iron sorption capacity in aqueous solution of őĪ-hydroxypolycarboxylic acids was less than the unmodified films (56.7 mg/g). This study demonstrated that Ch-GalA Ge films had a 34% decrease in iron ions sorption capacity (37.5 mg/g), supporting the hypothesis that sorption occurred by a mechanism of electrostatic interactions, leading to the formation of ternary chitosan-iron ions citrate complexes. Ch-based films crosslinked with genipin showed resistance to acidic media. However, genipin is a natural compound with low availability. Reducing sugars can be an alternative to genipin due to their capacity to form the crosslinking with chitosan through Maillard reaction. Thus, it was studied the feasibility to use glucose (Glc) and galacturonic acid (GalA) as crosslinkers. This study revealed that Ch-Glc and Ch-GalA films were insoluble in acidic media. Ch-Glc and Ch-GalA films showed also antioxidant activity due to antiradical activity and sorption capacity for iron ions in the presence of citric acid, allowing to conclude that chitosan-based films grafted with reducing sugars have potential to be used as an active material for food packaging as a preservation methodology. This PhD thesis allowed to achieve the knowledge regarding the mechanism by which chitosan can be used for the development of innovative films. Moreover, it was possible to further valorize the gathered knowledge, namely, how to modify the Ch-Ge films by the introduction of carboxylic function on the Ch-Ge films, thus rendering them resistance to acidic media. Moreover, Maillard reaction can be an effective approach to modify chitosan-based films with sugars as alternative to the Ch-Ge film.Os filmes de quitosana reticulados com genipina (Ch-Ge) permitem conservar vinhos brancos, contribuindo com notas de aroma positivas sem alterar a tipicidade do vinho. O processo de conserva√ß√£o est√° relacionado com a diminui√ß√£o do teor em ferro (50%) e cobre (16%), o que inibe as rea√ß√Ķes de oxida√ß√£o, bem como o crescimento de microrganismos. Na presen√ßa de filmes de Ch-Ge, os vinhos apresentam maior conte√ļdo de benzalde√≠do e furfural, o que permite pressupor que estes filmes podem promover as rea√ß√Ķes de Maillard. Assim, o principal objetivo desta tese de doutoramento foi proporcionar as bases cient√≠ficas que permitam compreender o impacto dos filmes de Ch-Ge na conserva√ß√£o dos vinhos. A forma√ß√£o de furfural e benzalde√≠do foi estudada em solu√ß√Ķes modelo de vinho na presen√ßa dos filmes de Ch-Ge. Os resultados mostraram que estes filmes promoveram a forma√ß√£o de furfural por desidrata√ß√£o da arabinose e de fenilacetalde√≠do e benzalde√≠do (alde√≠dos de Strecker) por degrada√ß√£o da fenilalanina. A forma√ß√£o de furfural foi demonstrada ocorrer devido aos grupos amina da quitosana. Apesar de ser poss√≠vel que os grupos alde√≠do do terminal redutor da quitosana reajam com amino√°cidos para formar os alde√≠dos de Strecker, o elevado conte√ļdo de benzalde√≠do n√£o pode ser justificado apenas pelo teor destes alde√≠dos. Foi demonstrado que os grupos alde√≠do da genipina que continuam a existir mesmo quando esta est√° ligada aos res√≠duos de glucosamina da quitosana, reagem com o grupo amina da fenilalanina, promovendo a forma√ß√£o de benzalde√≠do. Assim, os filmes Ch-Ge s√£o respons√°veis pela forma√ß√£o de alde√≠dos de Strecker, corroborando a ocorr√™ncia da rea√ß√£o de Maillard no vinho pela presen√ßa dos filmes. Solu√ß√Ķes modelo de genipina e 2-amino-2-desoxi-D-glucitol, mimetizando a unidade estrutural da quitosana sem o grupo redutor, permitiram verificar, por an√°lise por espectrometria de massa tandem com ioniza√ß√£o por electrospray, a forma√ß√£o de liga√ß√Ķes amida entre a genipina e os grupos amina do a√ß√ļcar aminado, confirmando a liga√ß√£o covalente entre a genipina e a quitosana, podendo manter os grupos alde√≠do da genipina livres. Para investigar em que extens√£o os filmes de Ch-Ge podem reter compostos vol√°teis, foram preparadas solu√ß√Ķes individuais contendo os filmes e compostos representativos de cinco fam√≠lias qu√≠micas, nomeadamente √°lcoois (hexanol), alde√≠dos (benzalde√≠do e hexanal), √°cidos carbox√≠licos (√°cido hexan√≥ico), √©steres (acetato de etilo e hexanoato de etilo) e cetonas (octan-3-ona). Foi poss√≠vel verificar que a capacidade de reten√ß√£o dos compostos vol√°teis pelos filmes de Ch-Ge aumentou com a hidrofobicidade das suas cadeias. No entanto, outros fen√≥menos, como eletrost√°tico, tamb√©m ocorreram, sendo preferencialmente retidos os √°cidos (54%, √°cido hexan√≥ico) e √©steres (38%, hexanoato de etilo), seguidos das cetonas (28%, octan-3-ona). Para perceber qual o mecanismo de redu√ß√£o dos i√Ķes ferro no vinho com a presen√ßa dos filmes de Ch-Ge, solu√ß√Ķes modelo de vinho foram preparadas contendo i√Ķes de ferro na presen√ßa dos filmes e acidificadas com √°cido tart√°rico ou √°cido clor√≠drico. Os resultados demonstraram que os filmes de Ch-Ge apenas apresentaram capacidade de sor√ß√£o para os i√Ķes ferro nas solu√ß√Ķes contendo √°cido tart√°rico. Com base nesta observa√ß√£o, foram estudados outros √°cidos carbox√≠licos, nomeadamente o √°cido ac√©tico, glic√≥lico, l√°ctico, succ√≠nico, m√°lico e c√≠trico. Verificou-se que os √°cidos őĪ hidroxipolicarbox√≠licos, como os √°cidos m√°lico e c√≠trico, levaram √† diminui√ß√£o dos i√Ķes ferro nestas solu√ß√Ķes, possivelmente devido √† forma√ß√£o de complexos tern√°rios por atra√ß√£o eletrost√°tica entre os grupos amina protonados da quitosana com a carga negativa dos complexos i√Ķes ferro hidroxipolicarboxilatos. A elucida√ß√£o do mecanismo de intera√ß√£o entre os i√Ķes ferro e a quitosana levou a que fossem desenvolvidos filmes √† base de quitosana modificada sem recurso √† presen√ßa de √°cidos őĪ-hidroxipolicarbox√≠licos que pudessem remover i√Ķes ferro de solu√ß√Ķes aquosas. A modifica√ß√£o da quitosana consistiu na introdu√ß√£o de grupos carbox√≠licos utilizando o anidrido diacetil-L-tart√°rico (Tartaril) e o √°cido galactur√≥nico (GalA). Foram preparados os filmes reticulados com genipina, obtendo-se filmes de N-tartarilCh-Ge e Ch-GalA-Ge. A capacidade de sor√ß√£o de i√Ķes ferro desses filmes foi avaliada em solu√ß√Ķes √°cidas sem a presen√ßa de √°cidos őĪ-hidroxipolicarbox√≠licos. No entanto, os filmes de N-tartarilCh-Ge e de Ch-GalA-Ge n√£o interagiram com os i√Ķes ferro, possivelmente pela ocorr√™ncia de reticula√ß√£o, o que leva √† aus√™ncia de grupos carbox√≠licos livres nos filmes modificados. Assim, os filmes em meio √°cido apenas possu√≠am grupos amina protonados o que leva a repuls√£o dos i√Ķes de ferro. Ambas as modifica√ß√Ķes qu√≠micas dos filmes levam a uma diminui√ß√£o do conte√ļdo de grupos amina comparativamente com os filmes de Ch-Ge, sendo expect√°vel que a capacidade de sor√ß√£o dos i√Ķes ferros numa solu√ß√£o de őĪ hidroxipolicarbox√≠licos seja inferior aos filmes n√£o modificados (Ch-Ge, 56,7 mg/g). Foi a avaliada a capacidade de sor√ß√£o dos i√Ķes ferro para os filmes de Ch-GalA-Ge numa solu√ß√£o aquosa de √°cido c√≠trico. Este estudo demonstrou que os filmes de Ch-Gal-Ge tiveram uma diminui√ß√£o de 34% na capacidade de sor√ß√£o (37,5 mg/g), corroborando a hip√≥tese de que a sor√ß√£o de ferro ocorreu por um mecanismo de intera√ß√Ķes eletrost√°ticas, levando √† forma√ß√£o de complexos tern√°rios de quitosana-i√Ķes ferro-citrato. Os filmes reticulados com genipina s√£o resistentes a meios √°cidos. No entanto, a genipina √© um composto natural com baixa disponibilidade. Os a√ß√ļcares redutores podem ser uma alternativa √† genipina, devido √† sua capacidade reticulante com a quitosana atrav√©s da rea√ß√£o de Maillard. Com este objetivo, foi estudada a viabilidade do uso de glucose (Glc) e √°cido galactur√≥nico (GalA) em alternativa √† Ge para produ√ß√£o de filmes. Este estudo revelou que os filmes Ch-Glc e Ch-GalA s√£o insol√ļveis em meio √°cido e possuem atividade antioxidante devido √† sua atividade antiradicalar, assim como a capacidade de sor√ß√£o de i√Ķes de ferro na presen√ßa de √°cido c√≠trico. Em resumo, os filmes √† base de quitosana reticulados com a√ß√ļcares redutores mostram potencial para serem usados como um material ativo em embalagens de alimentos. Esta tese de doutoramento permitiu conhecer o mecanismo pelo qual a quitosana pode ser utilizada para o desenvolvimento de filmes inovadores. Al√©m disso, foi poss√≠vel valorizar ainda mais o conhecimento acumulado, a saber, como modificar os filmes de Ch-Ge pela introdu√ß√£o da fun√ß√£o carbox√≠lica nos filmes de Ch-Ge, tornando-os resistentes a meios √°cidos. Al√©m disso, a rea√ß√£o de Maillard pode ser uma abordagem eficaz para modificar filmes √† base de quitosana com a√ß√ļcares como alternativa aos filmes Ch-Ge.Programa Doutoral em Qu√≠mica Sustent√°ve

    Estudo da remodelagem reversa miocárdica através da análise proteómica do miocárdio e do líquido pericárdico

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    Valve replacement remains as the standard therapeutic option for aortic stenosis patients, aiming at abolishing pressure overload and triggering myocardial reverse remodeling. However, despite the instant hemodynamic benefit, not all patients show complete regression of myocardial hypertrophy, being at higher risk for adverse outcomes, such as heart failure. The current comprehension of the biological mechanisms underlying an incomplete reverse remodeling is far from complete. Furthermore, definitive prognostic tools and ancillary therapies to improve the outcome of the patients undergoing valve replacement are missing. To help abridge these gaps, a combined myocardial (phospho)proteomics and pericardial fluid proteomics approach was followed, taking advantage of human biopsies and pericardial fluid collected during surgery and whose origin anticipated a wealth of molecular information contained therein. From over 1800 and 750 proteins identified, respectively, in the myocardium and in the pericardial fluid of aortic stenosis patients, a total of 90 dysregulated proteins were detected. Gene annotation and pathway enrichment analyses, together with discriminant analysis, are compatible with a scenario of increased pro-hypertrophic gene expression and protein synthesis, defective ubiquitinproteasome system activity, proclivity to cell death (potentially fed by complement activity and other extrinsic factors, such as death receptor activators), acute-phase response, immune system activation and fibrosis. Specific validation of some targets through immunoblot techniques and correlation with clinical data pointed to complement C3 ő≤ chain, Muscle Ring Finger protein 1 (MuRF1) and the dual-specificity Tyr-phosphorylation regulated kinase 1A (DYRK1A) as potential markers of an incomplete response. In addition, kinase prediction from phosphoproteome data suggests that the modulation of casein kinase 2, the family of IőļB kinases, glycogen synthase kinase 3 and DYRK1A may help improve the outcome of patients undergoing valve replacement. Particularly, functional studies with DYRK1A+/- cardiomyocytes show that this kinase may be an important target to treat cardiac dysfunction, provided that mutant cells presented a different response to stretch and reduced ability to develop force (active tension). This study opens many avenues in post-aortic valve replacement reverse remodeling research. In the future, gain-of-function and/or loss-of-function studies with isolated cardiomyocytes or with animal models of aortic bandingdebanding will help disclose the efficacy of targeting the surrogate therapeutic targets. Besides, clinical studies in larger cohorts will bring definitive proof of complement C3, MuRF1 and DYRK1A prognostic value.A substitui√ß√£o da v√°lvula a√≥rtica continua a ser a op√ß√£o terap√™utica de refer√™ncia para doentes com estenose a√≥rtica e visa a elimina√ß√£o da sobrecarga de press√£o, desencadeando a remodelagem reversa mioc√°rdica. Contudo, apesar do benef√≠cio hemodin√Ęmico imediato, nem todos os pacientes apresentam regress√£o completa da hipertrofia do mioc√°rdio, ficando com maior risco de eventos adversos, como a insufici√™ncia card√≠aca. Atualmente, os mecanismos biol√≥gicos subjacentes a uma remodelagem reversa incompleta ainda n√£o s√£o claros. Al√©m disso, n√£o dispomos de ferramentas de progn√≥stico definitivos nem de terapias auxiliares para melhorar a condi√ß√£o dos pacientes indicados para substitui√ß√£o da v√°lvula. Para ajudar a resolver estas lacunas, uma abordagem combinada de (fosfo)prote√≥mica e prote√≥mica para a caracteriza√ß√£o, respetivamente, do mioc√°rdio e do l√≠quido peric√°rdico foi seguida, tomando partido de bi√≥psias e l√≠quidos peric√°rdicos recolhidos em ambiente cir√ļrgico. Das mais de 1800 e 750 prote√≠nas identificadas, respetivamente, no mioc√°rdio e no l√≠quido peric√°rdico dos pacientes com estenose a√≥rtica, um total de 90 prote√≠nas desreguladas foram detetadas. As an√°lises de anota√ß√£o de genes, de enriquecimento de vias celulares e discriminativa corroboram um cen√°rio de aumento da express√£o de genes pro-hipertr√≥ficos e de s√≠ntese proteica, um sistema ubiquitina-proteassoma ineficiente, uma tend√™ncia para morte celular (potencialmente acelerada pela atividade do complemento e por outros fatores extr√≠nsecos que ativam death receptors), com ativa√ß√£o da resposta de fase aguda e do sistema imune, assim como da fibrose. A valida√ß√£o de alguns alvos espec√≠ficos atrav√©s de immunoblot e correla√ß√£o com dados cl√≠nicos apontou para a cadeia ő≤ do complemento C3, a Muscle Ring Finger protein 1 (MuRF1) e a dual-specificity Tyr-phosphoylation regulated kinase 1A (DYRK1A) como potenciais marcadores de uma resposta incompleta. Por outro lado, a predi√ß√£o de cinases a partir do fosfoproteoma, sugere que a modula√ß√£o da case√≠na cinase 2, a fam√≠lia de cinases do IőļB, a glicog√©nio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condi√ß√£o dos pacientes indicados para interven√ß√£o. Em particular, a avalia√ß√£o funcional de cardiomi√≥citos DYRK1A+/- mostraram que esta cinase pode ser um alvo importante para tratar a disfun√ß√£o card√≠aca, uma vez que os mi√≥citos mutantes responderam de forma diferente ao estiramento e mostraram uma menor capacidade para desenvolver for√ßa (tens√£o ativa). Este estudo levanta v√°rias hip√≥teses na investiga√ß√£o da remodelagem reversa. No futuro, estudos de ganho e/ou perda de fun√ß√£o realizados em cardiomi√≥citos isolados ou em modelos animais de banding-debanding da aorta ajudar√£o a testar a efic√°cia de modular os potenciais alvos terap√™uticos encontrados. Al√©m disso, estudos cl√≠nicos em coortes de maior dimens√£o trar√£o conclus√Ķes definitivas quanto ao valor de progn√≥stico do complemento C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin

    Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms

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    Biofilm may be a consortium of microbial species where the cells of microbes attach to both life form and inanimate surfaces inside a self-made matrix of extracellular polymeric substance (EPS). Biofilm matrix surrounding the polymicrobial environment makes them highly resistant to harsh conditions and antibacterial treatments. The two significant factors that differentiate planktonic from biofilm resident microbes are EPS containing a variety of macromolecules and a diffusible molecule for transferring signals known as quorum sensing (QS). Against this backdrop of microbial resistance and cell signaling, different approaches have been developed to interfere with the specific mechanisms of intracellular and extracellular targets that include herbal active compounds and synthetic nanoparticles. This chapter outlines the features of biofilm development and the approaches with the evidence that can be incorporated into clinical usage

    Desenvolvimento de testes genéticos por PCR em tempo-real para diagnóstico rápido de LHON e surdez

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    Mitochondrial cytopathies are a set of diseases caused by a disturbance in the cell energy production. Mitochondrial dysfunction impairs efficiency of the mitochondrial respiratory chain (MRC) and ATP production, affecting the organism‚Äôs energetic equilibrium. Pathogenic sequence variants in mitochondrial DNA (mtDNA) that lead to these pathologies are more frequent in tissues that need higher energy levels to function. The presented work looks into two such diseases: Leber‚Äôs Hereditary Optic Neuropathy and mitochondrial non-syndromic Hearing Loss (MNSHL). LHON is characterized by presence of genetic alterations in mtDNA, with three main primary pathogenic sequence variants existing, which represent 90-95% of LHON cases with an identified genetic cause: m.3460G>A, in ND1 subunit gene; m.11778G>A, in ND4 subunit gene; and m.14484T>C, in ND6 subunit gene. All of these are subunits of the MRC‚Äôs complex I. These mtDNA variations lead to mitochondrial dysfunction in complex I, creating ATP depletion, reactive oxygen species (ROS) increase and oxidative stress. LHON is commonly characterized by a sequential vision loss and, within 1 year of symptoms starting, 97% of patients with vision loss in one eye develop loss in the second. Therapy administration yields good outcomes, if done in a short-time span after first vision loss. It is essential to quickly and reliably scan for pathogenic sequence variants, in order to act timely and rescue function. Mitochondrial non-syndromic hearing loss and deafness (MNSHL) is characterized by sensorineural hearing loss (SNHL). This type of hearing loss, particularly when induced by aminoglycosides, has also three primary pathogenic sequence variants associated with ototoxicity: m.1494C>T and m.1555A>G, both in the MTRNR1 gene, and m.7445A>G, in the MTCO1 and MTTS1 genes. These are responsible for ATP depletion, an increase of ROS and oxidative stress, due to alterations in the mitochondrial ribosome or tRNA. In MNSHL, the cochlea is the affected tissue. With this disorder the principal modifier factor is the administration of aminoglycosides, a type of antibiotics, which trigger a cascade, that leads the individual permanently deaf. The best course of action is prevention, and to ensure clinical action is not dramatically slowed down, results that show whether administration is safe or not need to be quick. The aim of this work, for both diseases, is the development of a screening method characterized by fast and reliable approach for genetic assessment, to be used for clinical guidance, particularly in therapeutics. For LHON, the screening method is based on real-time PCR with High-Resolution Melting (HRM) analysis, for detection of the TOP-3 pathogenic sequence variants, by assessing the amplicon‚Äôs Tm. In this case, 94 samples were analyzed, including LHON suspected patients, relatives, other mitochondrial disease patients and healthy controls. All samples were previously classified by another method, having then been blinded before the performance of this work. For analysis, Real-Time PCR was run in triplicates, to allow for a more robust HRM analysis. The software had the ability to classify samples as different variants, wild-type or mutant; information which was then crossed with the previous classification of the sample to assess the success of the software classification. Samples were correctly assigned. This approach provides results in a quick fashion that guides clinical action in a timely fashion. The presence of other polymorphisms in the amplicons might be a hindrance to the robustness of the results provided by this technique and their effect on variant classification needs to be considered. For this, a predictive in-silico analysis was performed, regarding all described variants‚Äô presence in the sequences in analysis. Accordingly, an additional complementary method may be necessary for assurance of result‚Äôs specificity. For MNSHL, the screening method was also real-time PCR based, but this one was performed with Amplification-Refractory Mutation System (ARMS) primers, designed for the pathogenic sequence variants previously associated in literature for the MNSHL. Discrimination of results was done based on amplification in positive cases and lack of it in negative cases. This approach analyzed 32 samples, including MNSHL suspected patients, their relatives, other mitochondrial disease patients and healthy controls, but only results concerning the m.1555A>G were obtained timely. All samples were previously classified by another method, having then been blinded before performance of this work. For optimization, Real-Time PCR was run in duplicates, to increase robustness of analysis. The Real-Time software showed if samples amplified as wild-type or mutant, with classification following. This data was crossed with previous known classification of the samples to assess the success of the approach. All analyzed samples were correctly identified with this approach. However, two of the three pathogenic sequence variants did not achieve implementation within the timeframe necessary for their inclusion, namely m.1494C>T and m.7445A>G. The optimization of their screening was not possible and further work is necessary to optimize and implement the approach concerning the analysis for these variants. In conclusion, it was possible to implement an analysis method for LHON‚Äôs TOP-3 pathogenic sequence variants within 24h, which represents a big step in precision medicine for diagnosis of this disease. On the other hand, although the implementation was not concluded, a similar approach was started for MNSHL ‚Äď that, when concluded, will have an enormous impact in preventing aminoglycoside induced HL. This work represents a high impact scientific contribution in reverse translational research.As citopatias mitocondriais s√£o um conjunto de doen√ßas causadas por um dist√ļrbio na produ√ß√£o de energia celular. A disfun√ß√£o mitocondrial prejudica a efici√™ncia da cadeia respirat√≥ria mitocondrial (CRM) e a produ√ß√£o de ATP, afetando o equil√≠brio energ√©tico do organismo. As varia√ß√Ķes de sequ√™ncia patog√©nicas no DNA mitocondrial (mtDNA) que levam a estas patologias s√£o mais frequentes em tecidos que necessitam de maiores n√≠veis de energia para funcionar. O presente trabalho explora duas dessas doen√ßas: Neuropatia √≥tica heredit√°ria de Leber (LHON) e Surdez mitocondrial induzida por aminoglicos√≠deos. A LHON √© caracterizada pela presen√ßa de altera√ß√Ķes gen√©ticas do mtDNA, existindo tr√™s varia√ß√Ķes de sequ√™ncia patog√©nicas prim√°rias principais, que representam 90-95% de casos de LHON com identifica√ß√£o da causa gen√©tica: m.3460G>A, no gene que codifica a subunidade ND1; m.11778G>A, no gene que codifica a subunidade ND4; e m.14484T>C, no gene que codifica a subunidade ND6. Todas estas subunidades pertencem ao complexo I da CRM. Estas altera√ß√Ķes no mtDNA levam a disfun√ß√£o mitocondrial no complexo I, criando deple√ß√£o de ATP, aumento de esp√©cies reativas de oxig√©nio (ROS) e stresse oxidativo. A LHON √© comummente caracterizada pela perda sequencial de vis√£o e, 1 ano ap√≥s o in√≠cio dos sintomas, 97% dos casos com perda de vis√£o num olho desenvolvem perda de vis√£o no segundo. A administra√ß√£o de terapia produz bons resultados, quando realizada num curto per√≠odo de tempo ap√≥s a primeira perda de vis√£o. Assim, √© essencial pesquisar varia√ß√Ķes de sequ√™ncia patog√©nicas gen√©ticas de forma r√°pida e fi√°vel, para atuar rapidamente e recuperar a fun√ß√£o visual. A Surdez mitocondrial n√£o-sindr√≥mica (MNSHL), em particular a induzida por aminoglicos√≠deos, tem tamb√©m tr√™s muta√ß√Ķes principais associadas √† perda de audi√ß√£o: m.1494C>T e m.1555A>G, ambas no gene MTRNR1, e m.7445A>G, nos genes MTCO1 e MTTS1. Estas s√£o respons√°veis pela deple√ß√£o de ATP, aumento de ROS e stresse oxidativo, devido a altera√ß√Ķes no ribossoma ou no tRNA mitocondrial. Aqui, o tecido afetado √© a c√≥clea. Nesta doen√ßa, o fator modificador em destaque √© a administra√ß√£o de antibi√≥ticos de tipo aminoglicos√≠deos, que despoletam uma cascata de acontecimentos, levando √† surdez permanente. A melhor estrat√©gia passa pela preven√ß√£o, enquanto ao mesmo tempo se garante que a a√ß√£o cl√≠nica n√£o sofre atrasos. Desta forma, s√£o necess√°rios resultados r√°pidos, que demonstrem se a administra√ß√£o ser√° segura ou n√£o. O objetivo deste trabalho, para ambas as doen√ßas, √© o desenvolvimento de um m√©todo de screening, caracterizado por uma abordagem r√°pida e fi√°vel, usado para guiar a decis√£o cl√≠nica, particularmente na terap√™utica. Para a LHON, o m√©todo de screening √© baseado em PCR em tempo-real com an√°lise de High-Resolution Melting (HRM), para dete√ß√£o das variantes patog√©nicas TOP-3, avaliando as Tm dos amplicons. Neste caso, foram analisadas 94 amostras, incluindo doentes com suspeita de LHON, familiares, outros doentes com suspeita de outra doen√ßa mitocondrial e controlos saud√°veis. Todas as amostras foram previamente classificadas por outro m√©todo, tendo sido sujeitas a anonimiza√ß√£o antes da realiza√ß√£o do trabalho. Para a an√°lise, a PCR em tempo-real foi realizada em triplicados, para permitir uma an√°lise de HRM mais robusta. O software teve a capacidade de classificar amostras como diferentes variantes, ou seja, normal ou mutante. Esta informa√ß√£o foi cruzada com as classifica√ß√Ķes previamente existentes para avaliar o sucesso da classifica√ß√£o pelo software. As amostras foram corretamente classificadas. Esta abordagem fornece resultados de forma r√°pida, podendo guiar a a√ß√£o cl√≠nica em tempo √ļtil. A presen√ßa de outros polimorfismos nos amplicons poder√£o obstruir a robustez dos resultados fornecidos por esta t√©cnica e o seu efeito na classifica√ß√£o de variantes precisa de ser considerado. Por esta raz√£o, foi realizada uma an√°lise de previs√£o in-silico, considerando a presen√ßa de todas as variantes descritas. Nesse sentido, pode ser necess√°rio um m√©todo complementar de an√°lise para assegurar a especificidade dos resultados. Para a Surdez mitocondrial n√£o-sindr√≥mica, o m√©todo de screening baseou-se tamb√©m na PCR em tempo-real, mas foi realizada com primers de Amplification-Refractory mutation system (ARMS), desenhados para as variantes de sequ√™ncia patog√©nicas associadas √† MNSHL induzida por aminoglicos√≠deos, previamente descritas na literatura para esta doen√ßa. A discrimina√ß√£o de resultados foi feita com base na presen√ßa/aus√™ncia de amplifica√ß√£o para cada variante. Foram analisadas 32 amostras com esta abordagem, incluindo doentes com suspeita de MNSHL, seus familiares, doentes com suspeita de outra doen√ßa mitocondrial e controlos saud√°veis, mas apenas foram obtidos resultados em tempo √ļtil para a m.1555A>G. Todas as amostras tinham sido previamente classificadas por outro m√©todo, tendo sido anonimizadas antes da realiza√ß√£o do trabalho. Para a otimiza√ß√£o, a PCR em tempo-real foi realizada em duplicados, aumentando a robustez da an√°lise. O software de tempo-real mostrou quais as amostras que amplificaram como normais ou mutantes, permitindo a classifica√ß√£o das mesmas. Os dados foram comparados com as classifica√ß√Ķes previamente conhecidas, para avaliar o sucesso da abordagem em estudo. Todas as amostras em an√°lise foram corretamente identificadas. No entanto, duas das tr√™s variantes patog√©nicas n√£o foram implementadas em tempo √ļtil para inclus√£o neste trabalho. Para a m.1494C>T e a m.7445A>G, a otimiza√ß√£o n√£o foi poss√≠vel, e ser√° necess√°rio trabalho adicional no futuro, para a implementa√ß√£o da an√°lise destas variantes. Em conclus√£o, foi poss√≠vel implementar um m√©todo da an√°lise das variantes gen√©ticas TOP-3 da LHON em 24h, o que representa um grande passo na medicina de precis√£o para diagn√≥stico desta doen√ßa. Por outro lado, apesar de n√£o ter sido conclu√≠da a implementa√ß√£o, iniciou-se uma abordagem semelhante para a MNSHL ‚Äď que, quando for conclu√≠da, ter√° um enorme impacto para evitar a perda auditiva por exposi√ß√£o a aminoglicos√≠deos. Este trabalho representa uma contribui√ß√£o cient√≠fica de alto impacto na investiga√ß√£o translacional reversa.O Laborat√≥rio de Biomedicina Mitocondrial e Teran√≥stica recebeu apoio financeiro da Santhera Pharmaceuticals que permitiu implementa√ß√£o do projeto nacional ‚ÄúInvestiga√ß√£o Translacional Epidemiol√≥gica, Bigen√≥mica e Funcional nas Atrofias √ďpticas‚ÄĚ (IP Professora Doutora Manuela Grazina). Apoio financeiro do CNC.IBILI no √Ęmbito do Plano Estrat√©gico UID/NEU/04539/2019.Mestrado em Biologia Aplicad

    Investigation of a Histidine-Based Probe for the Exploration of Proteomes

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    Leishmaniasis is a neglected tropical disease which affects 0.7-1 million people per year. Current chemotherapies for leishmaniasis are toxic with long treatment times and reports of increasing resistance, which stresses the importance of this research area. Inositol phosphorylceramide synthase is a membrane bound enzyme that has no direct human homologue, which converts ceramide to inositol phosphorylceramide through the action of a highly conserved HHD catalytic triad. An ideal method to study this enzyme further would be through activity-based protein profiling, however, there are currently no activity-based probes reported that reacts with this type of active site. Therefore, an activity-based probe was designed based on the structure of diethyl pyrocarbonate, a compound known to bind covalently to active site histidine residues. The synthesised activity-based probe was shown to inhibit Leishmania major inositol phosphorylceramide synthase in a simple assay. In addition, the probe was shown to selectively bind to the active site histidine residue in two pure enzyme models; one of which has the same catalytic triad as inositol phosphorylceramide synthase, and the other was an acid base active site histidine residue. Further, this activity-based probe was able to isolate an overexpressed enzyme in the lysate of Escherichia coli as well as bind to intrinsic proteins. Following the function validation of the activity-based probe, preliminary work was started in Leishmania to isolate proteins identify expressed enzymes
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