73 research outputs found

    Monte Carlo studies of two-dimensional polymerÔÇôsolvent systems

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    <div><p>Allergic bronchopulmonary aspergillosis (ABPA) in asthma is a severe, life-affecting disease that potentially affects over 4.8 million people globally. In the UK, ABPA is predominantly caused by the fungus <i>Aspergillus fumigatus</i>. Phagocytosis is important in clearance of this fungus, and Early Endosome Antigen 1 (<i>EEA1</i>) has been demonstrated to be involved in phagocytosis of fungi. We sought to investigate the role of <i>EEA1</i> mutations and phagocytosis in ABPA. We used exome sequencing to identify variants in <i>EEA1</i> associated with ABPA. We then cultured monocyte-derived macrophages (MDMs) from 17 ABPA subjects with <i>A</i>. <i>fumigatus</i> conidia, and analyzed phagocytosis and phagolysosome acidification in relation to the presence of these variants. We found that variants in <i>EEA1</i> were associated with ABPA and with the rate of phagocytosis of <i>A</i>. <i>fumigatus</i> conidia and the acidification of phagolysosomes. MDMs from ABPA subjects carrying the disease associated genotype showed increased acidification and phagocytosis compared to those from ABPA subjects carrying the non-associated genotypes or healthy controls.The identification of ABPA-associated variants in EEA that have functional effects on MDM phagocytosis and phagolysosome acidification of <i>A</i>. <i>fumigatus</i> conidia revolutionizes our understanding of susceptibility to this disease, which may in future benefit patients by earlier identification or improved treatments. We suggest that the increased phagocytosis and acidification observed demonstrates an over-active MDM profile in these patients, resulting in an exaggerated cellular response to the presence of <i>A</i>. <i>fumigatus</i> in the airways.</p></div

    SM with influence on virulence of fungi in the respective host system.

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    <p>Animal cell: <b>Gliotoxin</b> acts on the mitochondrial protein Bak, which leads to apoptosis and inhibits the activation of NF╬║B through blocking proteasomal activity; <b>DHN-melanin</b> from <i>A. fumigatus</i> inhibits vH<sup>+</sup>-ATPase activity and therefore acidification of the phagolysosome, which counteracts killing of the conidia. Fungi: <b>Siderophores</b> are released by the fungus to ensure iron availability; <b>DHN-melanin</b> from plant pathogens is crucial for appressorium formation and counteracting reactive oxygen species (ROS) produced by the plant through hypersensitive response (HR) to prevent the spread of fungal infection; <b>DOPA-melanin</b> produced by <i>C. neoformans</i> from external precursors is an important part of the fungal capsule. Plant cell: <b>Cytochalasins</b> block cytokinesis; <b>T-Toxin</b> inhibits mitochondrial energy production; <b>Fumonisin</b>, <b>AAL-Toxin</b>, and <b>Cyperin</b> affect the membrane integrity; <b>Auxin</b> and <b>Gibberellins</b> act as phytohormones and alter transcription activity; <b>HC-Toxin</b> inhibits histone deacetylase; after light-driven activation both <b>Cercosporin</b> and <b>Elsinochromes</b> produce ROS, which damage the cell; <b>Sirodesmin</b> induces ROS and the formation of protein-conjugates; <b>Tentoxin</b> inhibits chloroplastidial energy production; <b>Fusicoccin</b> mediates irreversible stomata opening; <b>Beticolin</b> forms pores in the membrane and leads to leakage of the cell.</p

    Effects of chloroquine treatment and phagosomal acidification on <i>A</i>.<i>fumigatus</i> infection in an apoptotic cell.

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    <p>The acidified pH of the apoptotic cell in the presence of chloroquine was maintained after infection. As it can be concluded, <i>A</i>.<i>fumigatus</i> only interferes with pH if the phagolysosme stays intact.</p

    Spectrum of the pH at given time points (6 h and 10 h p.i.) in apoptotic and non-apoptotic infected monocytes.

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    <p>The dashed lines (shown in blue and green) are the spectra of pH in wild-type (wt.) and mutant infection in non-apoptotic monocyte at 6 h p.i. when their pH showed approximately similar values (6.3 to 6.5). The constant lines represent the wild-type or mutant infections in apoptotic monocyte (treated with STS) comparing two time points of 6 and 10 h p.i. The most acidic pH (less than 4) was recorded 10 h p.i. in the apoptotic monocyte infected with mutant whereas at the same time point the apoptotic monocyte infected with wild-type showed a higher pH around 5.3.Regardless of the type of infection, the signals related to acidic pH in non-apoptotic cells were generally less intense than in apoptotic cells. 10 h p.i., the apoptotic cells infected by melanin-free <i>pksP</i> mutant conidia were far more acidic compared to cells containing wild-type conidia.</p

    Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters <i>In Vivo</i> during Interaction of the Pathogenic Fungus <i>Aspergillus fumigatus</i> with Human Monocytes

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    <div><p>Hyperspectral imaging (HSI) is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus <i>Aspergillus fumigatus</i> conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised <i>A</i>. <i>fumigatus</i> conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of <i>A</i>. <i>fumigatus</i> is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell.</p></div

    Opposed Effects of Enzymatic Gliotoxin <i>N</i>- and <i>S</i>ÔÇĹMethylations

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    Gliotoxin (<b>1</b>), a virulence factor of the human pathogenic fungus Aspergillus fumigatus, is the prototype of epipoly┬ş(thiodioxopiperazine) (ETP) toxins. Here we report the discovery and functional analysis of two methyl transferases (MTs) that play crucial roles for ETP toxicity. Genome comparisons, knockouts, and in vitro enzyme studies identified a new <i>S</i>-adenosyl-l-methionine-dependent <i>S</i>-MT (TmtA) that is, surprisingly, encoded outside the <i>gli</i> gene cluster. We found that TmtA irreversibly inactivates ETP by <i>S</i>-alkylation and that this detoxification strategy appears to be not only limited to ETP producers. Furthermore, we unveiled that GliN functions as a freestanding amide <i>N</i>-MT. GliN-mediated amide methylation confers stability to ETP, damping the spontaneous formation of tri- and tetrasulfides. In addition, enzymatic <i>N</i>-alkylation constitutes the last step in gliotoxin biosynthesis and is a prerequisite for the cytotoxicity of the molecule. Thus, these specialized alkylating enzymes have dramatic and fully opposed effects: complete activation or inactivation of the toxin

    Kinetics of phagolysosomal pH upon infection.

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    <p><b>(A)</b> Phagosomal pH in an apoptotic monocyte containing labelled wild-type conidia in comparison to an apoptotic monocyte carrying <i>pksP</i> mutant conidia. <b>(B)</b> Data represent the mean <sub>+</sub> SD from three experiments. <b>(C)</b> Phagosomal pH in the survived apoptotic monocyte infected with labelled wild-type conidia. <b>(D)</b> Data represent the mean <sub>+</sub> SD of cytosolic pH from three experiments.</p

    Effect of bafilomycin A1 treatment and inhibition of phagosomal acidification.

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    <p>Despite the inhibitory effect of bafilomycin on phagosomes-lysosome degradation, in the presence of melanin the engulfment of conidia and inhibition of intra-cellular acidification were not affected and the conidia were localised in the phagolysosome. Wild-type infection prevented acidification of phagosome, while the <i>pksP</i> mutant was not able to recover the acidic pH.</p

    Rewiring of the Austinoid Biosynthetic Pathway in Filamentous Fungi

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    Filamentous fungi produce numerous high-value natural products (NPs). The biosynthetic genes for NPs are normally clustered in the genome. A valuable NP class is represented by the insecticidal austinoids. We previously determined their biosynthesis in the fungus <i>Aspergillus calidoustus</i>. After further computational analysis looking into the austinoid gene clusters in two additional distantly related fungi, <i>Aspergillus nidulans</i> and <i>Penicillium brasilianum</i>, a rearrangement of the genes was observed that corresponded to the diverse austinoid derivatives produced by each strain. By advanced targeted combinatorial engineering using polycistronic expression of selected genes, we rewired the austinoid pathway in the fungus <i>A. nidulans</i>, which then produced certain compounds of interest under industrially favored conditions. This was possible by exploiting the presence of genes previously thought to be irrelevant. Our work shows that comparative analysis of genomes can be used to not only discover new gene clusters but unearth the hidden potential of known metabolic pathways

    Spectrum of the pH at given time points (6 h and 10 h p.i.) in apoptotic and non-apoptotic infected monocytes.

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    <p>The dashed lines (shown in blue and green) are the spectra of pH in wild-type (wt.) and mutant infection in non-apoptotic monocyte at 6 h p.i. when their pH showed approximately similar values (6.3 to 6.5). The constant lines represent the wild-type or mutant infections in apoptotic monocyte (treated with STS) comparing two time points of 6 and 10 h p.i. The most acidic pH (less than 4) was recorded 10 h p.i. in the apoptotic monocyte infected with mutant whereas at the same time point the apoptotic monocyte infected with wild-type showed a higher pH around 5.3.Regardless of the type of infection, the signals related to acidic pH in non-apoptotic cells were generally less intense than in apoptotic cells. 10 h p.i., the apoptotic cells infected by melanin-free <i>pksP</i> mutant conidia were far more acidic compared to cells containing wild-type conidia.</p
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