33,309 research outputs found

    Multiple functions and regulatory network of miR-150 in B lymphocyte-related diseases

    Get PDF
    MicroRNAs (miRNAs) play vital roles in the post-transcriptional regulation of gene expression. Previous studies have shown that miR-150 is a crucial regulator of B cell proliferation, differentiation, metabolism, and apoptosis. miR-150 regulates the immune homeostasis during the development of obesity and is aberrantly expressed in multiple B-cell-related malignant tumors. Additionally, the altered expression of MIR-150 is a diagnostic biomarker of various autoimmune diseases. Furthermore, exosome-derived miR-150 is considered as prognostic tool in B cell lymphoma, autoimmune diseases and immune-mediated disorders, suggesting miR-150 plays a vital role in disease onset and progression. In this review, we summarized the miR-150-dependent regulation of B cell function in B cell-related immune diseases

    Pollution-induced community tolerance in freshwater biofilms ÔÇô from molecular mechanisms to loss of community functions

    Get PDF
    Exposure to herbicides poses a threat to aquatic biofilms by affecting their community structure, physiology and function. These changes render biofilms to become more tolerant, but on the downside community tolerance has ecologic costs. A concept that addresses induced community tolerance to a pollutant (PICT) was introduced by Blanck and W├Ąngberg (1988). The basic principle of the concept is that microbial communities undergo pollution-induced succession when exposed to a pollutant over a long period of time, which changes communities structurally and functionally and enhancing tolerance to the pollutant exposure. However, the mechanisms of tolerance and the ecologic consequences were hardly studied up to date. This thesis addresses the structural and functional changes in biofilm communities and applies modern molecular methods to unravel molecular tolerance mechanisms. Two different freshwater biofilm communities were cultivated for a period of five weeks, with one of the communities being contaminated with 4 ╬╝g L-1 diuron. Subsequently, the communities were characterized for structural and functional differences, especially focusing on their crucial role of photosynthesis. The community structure of the autotrophs was assessed using HPLC-based pigment analysis and their functional alterations were investigated using Imaging-PAM fluorometry to study photosynthesis and community oxygen profiling to determine net primary production. Then, the molecular fingerprints of the communities were measured with meta-transcriptomics (RNA-Seq) and GC-based community metabolomics approaches and analyzed with respect to changes in their molecular functions. The communities were acute exposed to diuron for one hour in a dose-response design, to reveal a potential PICT and uncover related adaptation to diuron exposure. The combination of apical and molecular methods in a dose-response design enabled the linkage of functional effects of diuron exposure and underlying molecular mechanisms based on a sensitivity analysis. Chronic exposure to diuron impaired freshwater biofilms in their biomass accrual. The contaminated communities particularly lost autotrophic biomass, reflected by the decrease in specific chlorophyll a content. This loss was associated with a change in the molecular fingerprint of the communities, which substantiates structural and physiological changes. The decline in autotrophic biomass could be due to a primary loss of sensitive autotrophic organisms caused by the selection of better adapted species in the course of chronic exposure. Related to this hypothesis, an increase in diuron tolerance has been detected in the contaminated communities and molecular mechanisms facilitating tolerance have been found. It was shown that genes of the photosystem, reductive-pentose phosphate cycle and arginine metabolism were differentially expressed among the communities and that an increased amount of potential antioxidant degradation products was found in the contaminated communities. This led to the hypothesis that contaminated communities may have adapted to oxidative stress, making them less sensitive to diuron exposure. Moreover, the photosynthetic light harvesting complex was altered and the photoprotective xanthophyll cycle was increased in the contaminated communities. Despite these adaptation strategies, the loss of autotrophic biomass has been shown to impair primary production. This impairment persisted even under repeated short-term exposure, so that the tolerance mechanisms cannot safeguard primary production as a key function in aquatic systems.:1. The effect of chemicals on organisms and their functions .............................. 1 1.1 Welcome to the anthropocene .......................................................................... 1 1.2 From cellular stress responses to ecosystem resilience ................................... 3 1.2.1 The individual pursuit for homeostasis ....................................................... 3 1.2.2 Stability from diversity ................................................................................. 5 1.3 Community ecotoxicology - a step forward in monitoring the effects of chemical pollution? ................................................................................................................. 6 1.4 Functional ecotoxicological assessment of microbial communities ................... 9 1.5 Molecular tools ÔÇô the key to a mechanistic understanding of stressor effects from a functional perspective in microbial communities? ...................................... 12 2. Aims and Hypothesis ......................................................................................... 14 2.1 Research question .......................................................................................... 14 2.2 Hypothesis and outline .................................................................................... 15 2.3 Experimental approach & concept .................................................................. 16 2.3.1 Aquatic freshwater biofilms as model community ..................................... 16 2.3.2 Diuron as model herbicide ........................................................................ 17 2.3.3 Experimental design ................................................................................. 18 3. Structural and physiological changes in microbial communities after chronic exposure - PICT and altered functional capacity ................................................. 21 3.1 Introduction ..................................................................................................... 21 3.2 Methods .......................................................................................................... 23 3.2.1 Biofilm cultivation ...................................................................................... 23 3.2.2 Dry weight and autotrophic index ............................................................. 23 3.2.4 Pigment analysis of periphyton ................................................................. 23 3.2.4.1 In-vivo pigment analysis for community characterization ....................... 24 3.2.4.2 In-vivo pigment analysis based on Imaging-PAM fluorometry ............... 24 3.2.4.3 In-vivo pigment fluorescence for tolerance detection ............................. 26 3.2.4.4 Ex-vivo pigment analysis by high-pressure liquid-chromatography ....... 27 3.2.5 Community oxygen metabolism measurements ....................................... 28 3.3 Results and discussion ................................................................................... 29 3.3.1 Comparison of the structural community parameters ............................... 29 3.3.2 Photosynthetic activity and primary production of the communities after selection phase ................................................................................................. 33 3.3.3 Acquisition of photosynthetic tolerance .................................................... 34 3.3.4 Primary production at exposure conditions ............................................... 36 3.3.5 Tolerance detection in primary production ................................................ 37 3.4 Summary and Conclusion ........................................................................... 40 4. Community gene expression analysis by meta-transcriptomics ................... 41 4.1 Introduction to meta-transcriptomics ............................................................... 41 4.2. Methods ......................................................................................................... 43 4.2.1 Sampling and RNA extraction................................................................... 43 4.2.2 RNA sequencing analysis ......................................................................... 44 4.2.3 Data assembly and processing................................................................. 45 4.2.4 Prioritization of contigs and annotation ..................................................... 47 4.2.5 Sensitivity analysis of biological processes .............................................. 48 4.3 Results and discussion ................................................................................... 48 4.3.1 Characterization of the meta-transcriptomic fingerprints .......................... 49 4.3.2 Insights into community stress response mechanisms using trend analysis (DRomicÔÇÖs) ......................................................................................................... 51 4.3.3 Response pattern in the isoform PS genes .............................................. 63 4.5 Summary and conclusion ................................................................................ 65 5. Community metabolome analysis ..................................................................... 66 5.1 Introduction to community metabolomics ........................................................ 66 5.2 Methods .......................................................................................................... 68 5.2.1 Sampling, metabolite extraction and derivatisation................................... 68 5.2.2 GC-TOF-MS analysis ............................................................................... 69 5.2.3 Data processing and statistical analysis ................................................... 69 5.3 Results and discussion ................................................................................... 70 5.3.1 Characterization of the metabolic fingerprints .......................................... 70 5.3.2 Difference in the metabolic fingerprints .................................................... 71 5.3.3 Differential metabolic responses of the communities to short-term exposure of diuron ............................................................................................................ 73 5.4 Summary and conclusion ................................................................................ 78 6. Synthesis ............................................................................................................. 79 6.1 Approaches and challenges for linking molecular data to functional measurements ...................................................................................................... 79 6.2 Methods .......................................................................................................... 83 6.2.1 Summary on the data ............................................................................... 83 6.2.2 Aggregation of molecular data to index values (TELI and MELI) .............. 83 6.2.3 Functional annotation of contigs and metabolites using KEGG ................ 83 6.3 Results and discussion ................................................................................... 85 6.3.1 Results of aggregation techniques ........................................................... 85 6.3.2 Sensitivity analysis of the different molecular approaches and endpoints 86 6.3.3 Mechanistic view of the molecular stress responses based on KEGG functions ............................................................................................................ 89 6.4 Consolidation of the results ÔÇô holistic interpretation and discussion ............... 93 6.4.1 Adaptation to chronic diuron exposure - from molecular changes to community effects.............................................................................................. 93 6.4.2 Assessment of the ecological costs of Pollution-induced community tolerance based on primary production ............................................................. 94 6.5 Outlook ............................................................................................................ 9

    The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D

    Get PDF
    RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses

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

    Get PDF
    UFMylation involves the covalent modification of substrate proteins with UFM1 (UbiquitinÔÇÉfold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ERÔÇÉbound ribosomes and activates C53ÔÇÉmediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8ÔÇÉinteracting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIMÔÇÉmediated 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 UFMylationÔÇÉdependent fineÔÇÉtuning of C53ÔÇÉmediated autophagy activation

    JAZF1: A metabolic actor subunit of the NuA4/TIP60 chromatin modifying complex

    Get PDF
    The multisubunit NuA4/TIP60 complex is a lysine acetyltransferase, chromatin modifying factor and gene co-activator involved in diverse biological processes. The past decade has seen a growing appreciation for its role as a metabolic effector and modulator. However, molecular insights are scarce and often contradictory, underscoring the need for further mechanistic investigation. A particularly exciting route emerged with the recent identification of a novel subunit, JAZF1, which has been extensively linked to metabolic homeostasis. This review summarizes the major findings implicating NuA4/TIP60 in metabolism, especially in light of JAZF1 as part of the complex

    Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities

    Get PDF
    Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectivelyÔÇöboth modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1ÔÇôB3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study

    Molecular diagnosis of Mycoplasma bovis

    Get PDF
    Disserta├ž├úo de Mestrado Integrado em Medicina Veterin├íriaMycoplasma bovis is a bacteria responsible for different disease presentations in cattle, such as pneumonia, mastitis, otitis, genital disorders, keratoconjuntivitis and arthritis, presently considered as one of the major emerging pathogens affecting cattle. Until this day, it is responsible for losses in animal production of over 150 million euros across Europe. The pathogenesis of Mycoplasma-associated diseases is multifactorial and the highly variable surface lipoproteins allows a fast and efficient dissemination of M. bovis within the host and the herd. Due to its high antigenic plasticity, its ability to survive within multiple host cells and the capacity to establish multiple synergistic interactions with other pathogens, makes M. bovis and associated infections are a major challenge in Veterinary Medicine, since the vaccine is not efficient and antibiotics are almost inefficient. This study aims at developing and validating a quantitative PCR protocol for the diagnosis of M. bovis. 93 milk samples, from 5 different Portuguese farms, were collected, processed and each oneÔÇÖs DNA extracted to be analyzed through a qPCR method targeting the uvrC and uvrC2024 genes. Given the percentage of positivity, which was high, the conclusion we can take from the study is that there is still work to do, in terms of establishing a uniformed practice to tackle the wide presence of M. bovis in farms.RESUMO - Diagn├│stico Molecular de M. bovis - Mycoplasma bovis ├ę uma bact├ęria cuja infec├ž├úo pode ter diferentes apresenta├ž├Áes tais como pneumonia, mastite, otite, afec├ž├Áes genitais, queratoconjuntivite e artrites. ├ë um dos agentes considerados emergentes e que afecta a produ├ž├úo agropecu├íria, sendo respons├ível por perdas na ordem dos 150 milh├Áes de euros na Europa. Tem uma patog├ęnese multifactorial e as prote├şnas membranares ├á sua superf├şcie conferem uma variabilidade que permite uma r├ípida e eficiente dissemina├ž├úo no hospedeiro, e no rebanho. Esta variabilidade e capacidade de resistir ├á imunidade do hospedeiro, assim como as suas interac├ž├Áes sin├ęrgicas com outros agentes patog├ęnicos, tornam as infec├ž├Áes por M. bovis um obst├ículo dif├şcil de conter e ultrapassar na Medicina Veterin├íria, isto porque quer a antibioterapia, quer a vacina, n├úo s├úo eficientes. Este estudo procura desenvolver e contribuir para o estabelecimento de um protocolo de diagn├│stico para a detec├ž├úo de M. bovis. Vindas de 5 produ├ž├Áes portuguesas diferentes, 93 amostras foram processadas e analisadas atrav├ęs de um qPCR, com os genes uvrC e uvrC2024 como genes alvo. Dados os resultados, com uma positividade significativa pode-se considerar que ainda h├í trabalho pela frente em termos de estabelecer e uniformizar uma pr├ítica para combater a larga presen├ža de M. bovis nas explora├ž├Áes.N/

    Gene markers of dietary macronutrient composition and growth in the skeletal muscle of gilthead sea bream (Sparus aurata)

    Get PDF
    To increase our current knowledge on the nutritional regulation of growth and gene expression pattern in fish skeletal muscle, the effect of dietary macronutrient composition was assessed on digestibility, nutrient retention, growth performance, and the mRNA levels of key genes involved in functionality, growth and development of the skeletal muscle in gilthead sea bream (Sparus aurata). Long-term starvation decreased the expression of myogenic regulatory factors such as Myod2, Myf5, myogenin (Myog) and Myf6 in the skeletal muscle of S. aurata. The supply of high or medium protein, low carbohydrate diets enhanced growth parameters, feed efficiency ratio, feed conversion ratio and significantly upregulated myod2. However, the supply of low protein, high carbohydrate diets restricted growth and stimulated the mRNA levels of myostatin, while downregulated follistatin (fst), igf1, mtor and rps6. Microarray analysis revealed igfals, tnni2, and gadd45a as gene markers upregulated by diets enriched with protein, lipids and carbohydrates, respectively. The results of the present study show that in addition to myod2, fst, igf1, mtor and rps6, the expression levels of igfals, tnni2 and remarkably gadd45a in the skeletal muscle can be used as markers to evaluate the effect of dietary macronutrient changes on fish growth and muscle development in S. aurata

    Estudo da remodelagem reversa mioc├írdica atrav├ęs da an├ílise prote├│mica do mioc├írdio e do l├şquido peric├írdico

    Get PDF
    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 ╬▓ 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 I╬║B 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├ž├úo da v├ílvula a├│rtica continua a ser a op├ž├úo terap├¬utica de refer├¬ncia para doentes com estenose a├│rtica e visa a elimina├ž├úo da sobrecarga de press├úo, desencadeando a remodelagem reversa mioc├írdica. Contudo, apesar do benef├şcio hemodin├ómico imediato, nem todos os pacientes apresentam regress├úo completa da hipertrofia do mioc├írdio, ficando com maior risco de eventos adversos, como a insufici├¬ncia card├şaca. Atualmente, os mecanismos biol├│gicos subjacentes a uma remodelagem reversa incompleta ainda n├úo s├úo claros. Al├ęm disso, n├úo dispomos de ferramentas de progn├│stico definitivos nem de terapias auxiliares para melhorar a condi├ž├úo dos pacientes indicados para substitui├ž├úo da v├ílvula. Para ajudar a resolver estas lacunas, uma abordagem combinada de (fosfo)prote├│mica e prote├│mica para a caracteriza├ž├úo, respetivamente, do mioc├írdio e do l├şquido peric├írdico foi seguida, tomando partido de bi├│psias e l├şquidos peric├írdicos recolhidos em ambiente cir├║rgico. Das mais de 1800 e 750 prote├şnas identificadas, respetivamente, no mioc├írdio e no l├şquido peric├írdico dos pacientes com estenose a├│rtica, um total de 90 prote├şnas desreguladas foram detetadas. As an├ílises de anota├ž├úo de genes, de enriquecimento de vias celulares e discriminativa corroboram um cen├írio de aumento da express├úo de genes pro-hipertr├│ficos e de s├şntese proteica, um sistema ubiquitina-proteassoma ineficiente, uma tend├¬ncia para morte celular (potencialmente acelerada pela atividade do complemento e por outros fatores extr├şnsecos que ativam death receptors), com ativa├ž├úo da resposta de fase aguda e do sistema imune, assim como da fibrose. A valida├ž├úo de alguns alvos espec├şficos atrav├ęs de immunoblot e correla├ž├úo com dados cl├şnicos apontou para a cadeia ╬▓ 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├ž├úo de cinases a partir do fosfoproteoma, sugere que a modula├ž├úo da case├şna cinase 2, a fam├şlia de cinases do I╬║B, a glicog├ęnio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condi├ž├úo dos pacientes indicados para interven├ž├úo. Em particular, a avalia├ž├úo funcional de cardiomi├│citos DYRK1A+/- mostraram que esta cinase pode ser um alvo importante para tratar a disfun├ž├úo card├şaca, uma vez que os mi├│citos mutantes responderam de forma diferente ao estiramento e mostraram uma menor capacidade para desenvolver for├ža (tens├úo ativa). Este estudo levanta v├írias hip├│teses na investiga├ž├úo da remodelagem reversa. No futuro, estudos de ganho e/ou perda de fun├ž├úo realizados em cardiomi├│citos isolados ou em modelos animais de banding-debanding da aorta ajudar├úo a testar a efic├ícia de modular os potenciais alvos terap├¬uticos encontrados. Al├ęm disso, estudos cl├şnicos em coortes de maior dimens├úo trar├úo conclus├Áes definitivas quanto ao valor de progn├│stico do complemento C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin
    • ÔÇŽ
    corecore