Monte Carlo studies of two-dimensional polymer–solvent systems

<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.

<p>Animal cell: <b>Gliotoxin</b> acts on the mitochondrial protein Bak, which leads to apoptosis and inhibits the activation of NFκB through blocking proteasomal activity; <b>DHN-melanin</b> from <i>A. fumigatus</i> inhibits vH⁺-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

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

<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

<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

Rewiring of the Austinoid Biosynthetic Pathway in Filamentous Fungi

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.

<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

Principle of hyperspectral imaging (HSI) as a combination of conventional image processing and classical spectroscopy.

<p><b>(A)</b> Hyperspectral data cube includes a set of data that are layered on top of one another. Each pixel in the cube consists of an entire spectrum and the resulting image represents the corresponding wavelength band. Typically in hyperspectral imagery, the spatial information is collected in the X-Y plane and spectral information represented in the Z-direction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163505#pone.0163505.ref044" target="_blank">44</a>]. <b>(B)</b> HSI detector attached to side camera port of an inverted microscope. This combination comprises the imaging spectrograph with sensitive monochrome camera. The CCD camera attached to the spectrograph and the main body through a port. It records the spectral images in a time interval of microsecond-scale. The fluorescence light source is located in the back of camera segment and is adjustable to give an ample light intensity. The front DSLR camera obtains real images of the sample that is located on the heating stage. The filter shield contains combination of filters to detect different fluorescent probes with different excitation/emission rate. <b>(C)</b> Different spectral responses at distinct positions within the detection line. The monocyte is placed within the object field. The multiple fluorescence spectra of cell, conidium or their surroundings are simultaneously captured. The schematic shows a line of areas, and each line covers more than one pixel.</p

Mutations in <i>EEA1</i> are associated with allergic bronchopulmonary aspergillosis and affect phagocytosis of <i>Aspergillus fumigatus</i> by human macrophages - Fig 2

<p><b>Association of EEA1 genotype (A) and correlation of disease-associated alleles (B) with the percentage of phagolysosome acidification observed in MDMs after co-culture with <i>A</i>. <i>fumigatus</i>.</b> A) Median and interquartile ranges are shown. For the ABPA groups, each point represents an individual. For the healthy group, which is shown for interest, each point represents a replicate experiment using the same healthy subject. ABPA groups were compared by Mann-Whitney tests. Healthy groups were not compared as they only contained data for one individual, and are shown for interest only. B) Only ABPA subjects are shown, and each point represents an individual. Correlation was calculated using the Pearson R test.</p

<i>EEA1</i> mutations associated with ABPA.

<p><i>EEA1</i> mutations associated with ABPA.</p