33,862 research outputs found

    Holistic analysis of lysine acetylation in aquaculture pathogenic bacteria Vibrio alginolyticus under bile salt stress

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    Lysine acetylation modification is a dynamic and reversible post-translational modification, which plays an important role in the metabolism and pathogenicity of pathogenic bacteria. Vibrio alginolyticus is a common pathogenic bacterium in aquaculture, and bile salt can trigger the expression of bacterial virulence. However, little is known about the function of lysine acetylation in V. alginolyticus under bile salt stress. In this study, 1,315 acetylated peptides on 689 proteins were identified in V. alginolyticus under bile salt stress by acetyl-lysine antibody enrichment and high-resolution mass spectrometry. Bioinformatics analysis found that the peptides motif ****A*Kac**** and *******Kac****A* were highly conserved, and protein lysine acetylation was involved in regulating various cellular biological processes and maintaining the normal life activities of bacteria, such as ribosome, aminoacyl-tRNA biosynthesis, fatty acid metabolism, two-component system, and bacterial secretion system. Further, 22 acetylated proteins were also found to be related to the virulence of V. alginolyticus under bile salt stress through secretion system, chemotaxis and motility, and adherence. Finally, comparing un-treated and treated with bile salt stress lysine acetylated proteins, it was found that there were 240 overlapping proteins, and found amino sugar and nucleotide sugar metabolism, beta-Lactam resistance, fatty acid degradation, carbon metabolism, and microbial metabolism in diverse environments pathways were significantly enriched in bile salt stress alone. In conclusion, this study is a holistic analysis of lysine acetylation in V. alginolyticus under bile salt stress, especially many virulence factors have also acetylated

    Preparation of gelatin from broiler chicken stomach collagen

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    With the increasing consumption of poultry meat around the world, the use of chicken stomachs as a source of collagen is being offered. The objective of this study was to extract gelatin from the stomachs of broiler chickens and to estimate their gel strength, ash content, viscosity, gelling point, melting point, clarity and digestibility. An innovative biotechnological method based on the conditioning of collagen with a microbial endoproteinase (Protamex¬ģ) and hot-water extraction was used to control the chemical and thermal denaturation process of collagen to prepare gelatin. The experiments were planned using a Taguchi design, 2 factors at 3 levels; factor A for the amount of proteolytic enzyme (0.10, 0.15 and 0.20%) and factor B for the extraction temperature (55.0, 62.5 and 70.0 ¬įC). Data were statistically processed and analyzed at a significance level of 95%. The gelatin yield averaged 65 ¬Ī 8%; the gel strength ranged from 25 ¬Ī 1 to 439 ¬Ī 6 Bloom, the viscosity from 1.0 ¬Ī 0.4 to 3.40 ¬Ī 0.03 mPa¬∑s, gelling point from 14.0 ¬Ī 2.0 to 22.0 ¬Ī 2.0 ¬įC, melting point from 28.0 ¬Ī 1.0 to 37.0 ¬Ī 1.0 ¬įC. The digestibility of gelatin was 100.0% in all samples; the ash content was very low (0.44 ¬Ī 0.02‚Äď0.81 ¬Ī 0.02%). The optimal conditions for the enzymatic treatment of collagen from chicken stomachs were achieved at a higher temperature (70.0 ¬įC) and a lower amount of enzyme (0.10‚Äď0.15%). Conditioning chicken collagen with a microbial endoproteinase is an economically and environmentally friendly processing method, an alternative to the usual acid- or alkaline-based treatment that is used industrially. The extracted products can be used for food and pharmaceutical applications. ¬© 2022 by the authors.Univerzita Tom√°Ň°e Bati ve Zl√≠nńõ: IGA/FT/2022/00

    Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy

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    UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin‚Äźfold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER‚Äźbound ribosomes and activates C53‚Äźmediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8‚Äźinteracting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM‚Äźmediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation‚Äźdependent fine‚Äźtuning of C53‚Äźmediated autophagy activation

    Metabolites changes of a low-temperature and low-salt fermented Chinese kohlrabi during fermentation based on non-targeted metabolomic analysis

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    A low-temperature and low-salt industrially fermented Chinese kohlrabi (LSCK) was developed in this study, with the salt usage decreased by approximately 70% compared to the traditional high-salt fermented Chinese kohlrabi (HSCK). The differences in physicochemical properties, metabolites and overall flavors during LSCK fermented for 0, 45 and 90‚ÄČdays (d) were analyzed by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS), electronic nose (E-nose) and other techniques. The results showed that the total acid content increased significantly from 3.68 to 8.59‚ÄČg/kg. However, the protein content significantly decreased from 2.52/100 to 0.66‚ÄČg/100‚ÄČg. The number of lactic acid bacteria cells increased significantly from 3.69 to 4.46 log10CFU/g. Based on multivariate statistical analysis, 21, 14, and 15 differential metabolites were identified in the three treatment groups A1 (0 and 45‚ÄČdays), A2 (45 and 90‚ÄČdays), and A3 (0 and 90‚ÄČdays) respectively (VIP‚ÄČ>‚ÄČ1, p <‚ÄČ0.05, |log2FC|‚ÄČ‚Č•‚ÄČ1.1). Carbohydrates, sugar alcohols, amino acids and their derivatives were the main differential metabolites in the LSCKs fermented for different periods. Aminoacyl‚ąítRNA biosynthesis and glycine, serine and threonine metabolism pathways significantly correlated with the differential metabolites based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (p <‚ÄČ0.05). Furthermore, the overall odors were significantly different among the LSCKs with different fermentation periods, as detected by E-nose. The present study describes the change trend of metabolites during LSCK fermentation and elucidates important metabolic pathways in LSCK, providing a theoretical basis for the target regulation of functional metabolites in kohlrabi and the optimization of LSCK processing

    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

    Estudo de altera√ß√Ķes gen√©ticas no NAPRT e NAMPT em cancro

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    NAD+ is both a co-enzyme for oxidation-reduction reactions and a substrate for NAD+ -consuming enzymes and therefore, it is critical for many cellular processes. In mammalian cells, intracellular NAD+ can be synthesized through either de novo synthesis, from tryptophan, or via salvage pathways, from other precursors, such as NAM and NA which are converted by NAMPT and NAPRT, respectively. Changes in the NAD+ content in cells and tissues are linked with a wide variety of diseases, including cancer. A large proportion of cancer cases is still diagnosed only at an advanced stage and thus, there is a need for new affordable, specific, sensitive and non-invasive biomarkers. Salivary biomarkers can meet these criteria and thus, are promising tools in cancer screening, diagnostic and prognostic. The main objective of this work was to study genetic alterations in NAMPT and NAPRT in DNA samples from healthy donors and in samples from cancer patients. For this, DNA and RNA extraction from saliva samples was optimized, as a starting point to study this biofluid as a source for cancer biomarkers. The results from the bioinformatics analysis showed that the frequency of alterations in NAPRT and NAMPT genes is low in the cancer contexts investigated. Nevertheless, it is still necessary to further study the impact that these alterations might have. There is also a great need to investigate and optimize methods for saliva studies, in order to promote it as a liquid biopsy of regular use in clinical settings.O NAD+ funciona como coenzima em rea√ß√Ķes de oxida√ß√£o-redu√ß√£o e como um substrato para determinadas enzimas e tem, portanto, um papel cr√≠tico em muitos processos celulares. Nas c√©lulas de mam√≠feros, o NAD+ intracelular pode ser sintetizado atrav√©s da s√≠ntese de novo, a partir do triptofano, ou atrav√©s das vias de ‚Äúreciclagem‚ÄĚ, a partir de outros precursores, como NAM e NA, que s√£o convertidos por NAMPT e NAPRT, respetivamente. Altera√ß√Ķes no conte√ļdo de NAD+ em c√©lulas e tecidos est√£o relacionadas com uma ampla variedade de doen√ßas, incluindo cancro. Uma grande propor√ß√£o dos casos de tumores ainda √© diagnosticada j√° num estadio avan√ßado e, por isso, s√£o necess√°rios novos biomarcadores economicamente acess√≠veis, espec√≠ficos, sens√≠veis e n√£o invasivos. Os biomarcadores salivares conseguem cumprir esses crit√©rios e s√£o, assim, mol√©culas promissoras para o rastreio, diagn√≥stico e progn√≥stico de cancro. O principal objetivo deste trabalho foi estudar altera√ß√Ķes gen√©ticas no NAMPT e NAPRT em amostras de DNA de dadores saud√°veis e em amostras de pacientes com cancro. Para isto, foi otimizada a extra√ß√£o de DNA e RNA a partir de amostras de saliva, como ponto de partida para estudar este biofluido como fonte de biomarcadores de cancro. Os resultados da an√°lise bioinform√°tica mostraram que a frequ√™ncia de altera√ß√Ķes nos genes NAPRT e NAMPT √© baixa nos contextos de cancro investigados. Ainda assim, ser√£o necess√°rios mais estudos para analisar o impacto que estas altera√ß√Ķes poder√£o ter. H√° tamb√©m uma grande necessidade de investigar e otimizar m√©todos para estudos em saliva, a fim de promov√™-la como bi√≥psia l√≠quida de uso generalizado em ambiente cl√≠nico.Mestrado em Biomedicina Molecula

    Epigenetics : a catalyst of plant immunity against pathogens

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    The plant immune system protects against pests and diseases. The recognition of stress-related molecular patterns triggers localised immune responses, which are often followed by longer-lasting systemic priming and/or up-regulation of defences. In some cases, this induced resistance (IR) can be transmitted to following generations. Such transgenerational IR is gradually reversed in the absence of stress at a rate that is proportional to the severity of disease experienced in previous generations. This review outlines the mechanisms by which epigenetic responses to pathogen infection shape the plant immune system across expanding time scales. We review the cis- and trans-acting mechanisms by which stress-inducible epigenetic changes at transposable elements (TEs) regulate genome-wide defence gene expression and draw particular attention to one regulatory model that is supported by recent evidence about the function of AGO1 and H2A.Z in transcriptional control of defence genes. Additionally, we explore how stress-induced mobilisation of epigenetically controlled TEs acts as a catalyst of Darwinian evolution by generating (epi)genetic diversity at environmentally responsive genes. This raises questions about the long-term evolutionary consequences of stress-induced diversification of the plant immune system in relation to the long-held dichotomy between Darwinian and Lamarckian evolution
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