A Global and Targeted Proteomic Investigation of Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogen that can cause invasive disease in immunocompromised individuals and, less frequently, in immunocompetent hosts. Proteomic investigation of A. fumigatus has the potential to enable global analysis of protein expression, identify potential targets for vaccine or diagnostic tool development, and characterise system-wide responses to external stimuli. Implementation of a large-scale proteomic strategy lead to the identification of non-redundant proteins from mycelia (n = 390) and culture supernatants (n = 42) of A. fumigatus. Utilisation of MS-based proteomics facilitated the identification of proteins typically under-represented in 2D-PAGE proteome maps, including proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Pre-fractionation of complex protein samples, by gel-filtration or gold nanoparticle pre-incubation, demonstrated potential for reduction of sample complexity. Indirect identification of secondary metabolite cluster expression was achieved using a global MS-based proteomic approach, with proteins (n = 20) from LaeA-regulated clusters detected. Targeted immunoproteomics resulted in the identification of antigenic proteins (n = 25) from A. fumigatus, reactive with sera from healthy individuals, and characterisation of these proteins may shed light on the pathobiology of A. fumigatus. Mechanisms involved in the interaction of A. fumigatus with gliotoxin were also examined, using phenotypic analysis, comparative proteomics and metabolomics. Gliotoxin was observed to relieve H2O2-induced stress, in a dose-dependent manner (0 - 10 μg/ml) and this correlated with a significant increase in expression of the gliotoxin oxidoreductase GliT (p < 0.05). This indicates a role for gliotoxin, and potentially GliT, in relief of oxidative stress in A. fumigatus. Correspondingly, proteins associated with response to stress were observed to significantly decrease in expression in the co-addition condition, relative to H2O2 alone (p < 0.05). Comparative proteomic profiling of the gliotoxin-sensitive mutant, A. fumigatus ΔgliK, revealed perturbation of translation, the methyl cycle and the endoplasmic reticulum in response to gliotoxin. This informs on the mechanisms involved in gliotoxin-mediated toxicity and may apply to other gliotoxin-sensitive species. Loss of gliotoxin production in A. fumigatus ΔgliK correlated with significant elevation in intracellular ergothioneine levels (p < 0.001). This study describes the first identification of ergothioneine in A. fumigatus and represents a target for future redox investigations

Friendship features associated with college students\u27 friendship maintenance and dissolution following problems

This study examined the association between friendship features (i.e., support and resources, trust, similarity, common experiences, commitment, intimate communication, balance of costs and rewards, personal characteristics and behaviors, length of friendship, amount of time spent with friend, closeness of the friendship, and expected ease of finding a new friend) and friendship outcomes (maintenance vs. dissolution and overall quality) following college students\u27 problems with their pre-college and college friends. Reported causes of college students\u27 pre-college and college friendship problems and friendship dissolution were examined. In addition, links between experiencing problems with friends and adjustment were explored. Friendship features and problems were assessed through several written questionnaires. Pre-college friendships were more susceptible to problems and dissolution than were college friendships. Expected ease of finding a new friend predicted friendship quality, maintenance, and dissolution for pre-college and college friends. Commitment and length of friendship predicted friendship quality for both types of friends, and the balance of costs and rewards predicted outcomes for college friends. College students did not identify the same causes of friendship problems and endings; a lack of common experiences was most likely to be mentioned as causing actual friendship dissolution but unlikely to be identified as causing friendship problems. When asked to indicate how often various specific events contributed to friendship problems and dissolution, students indicated that many events were more frequent and more important in their pre-college friendships. Students who indicated that they frequently experienced many problems with their friends also reported higher levels of loneliness and homesickness and lower levels of social support. Links between adjustment measures and frequency of specific types of friendship problems were identified. For example, students who frequently had problems associated with similarity reported using alcohol infrequently and had relatively high grade point averages. Results generally suggest that college students\u27 friendship outcomes are similar to outcomes of other types of relationships. Implications for college personnel are discussed

Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl2-incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl2. Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products

Beyond Access: Bridging the Digital Divide

This paper describes the theoretical underpinnings of an ongoing research project that is examining the relationship between e-Democracy and the ‘digital divide’. The literature surrounding the Digital Divide is reviewed, and the importance of equitable physical access to ICTs in the drive to bridge the issue of social exclusion examined. It is argued that any discussion of the phenomenon of the digital divide must look beyond equitable physical access and take into consideration issues mentioned separately in the literature – ‘real access’, ‘reach’ and ‘socially responsible connectivity’

Label-efficient Contrastive Learning-based model for nuclei detection and classification in 3D Cardiovascular Immunofluorescent Images

Recently, deep learning-based methods achieved promising performance in nuclei detection and classification applications. However, training deep learning-based methods requires a large amount of pixel-wise annotated data, which is time-consuming and labor-intensive, especially in 3D images. An alternative approach is to adapt weak-annotation methods, such as labeling each nucleus with a point, but this method does not extend from 2D histopathology images (for which it was originally developed) to 3D immunofluorescent images. The reason is that 3D images contain multiple channels (z-axis) for nuclei and different markers separately, which makes training using point annotations difficult. To address this challenge, we propose the Label-efficient Contrastive learning-based (LECL) model to detect and classify various types of nuclei in 3D immunofluorescent images. Previous methods use Maximum Intensity Projection (MIP) to convert immunofluorescent images with multiple slices to 2D images, which can cause signals from different z-stacks to falsely appear associated with each other. To overcome this, we devised an Extended Maximum Intensity Projection (EMIP) approach that addresses issues using MIP. Furthermore, we performed a Supervised Contrastive Learning (SCL) approach for weakly supervised settings. We conducted experiments on cardiovascular datasets and found that our proposed framework is effective and efficient in detecting and classifying various types of nuclei in 3D immunofluorescent images.Comment: 11 pages, 5 figures, MICCAI Workshop Conference 202

Functional Investigation of Iron-Responsive Microsomal Proteins, including MirC, in Aspergillus fumigatus

The functionality of many microsome-associated proteins which exhibit altered abundance in response to iron limitation in Aspergillus fumigatus is unknown. Here, we generate and characterize eight gene deletion strains, and of most significance reveal that MirC (AFUA_2G05730) contributes to the maintenance of intracellular siderophore [ferricrocin (FC)] levels, augments conidiation, confers protection against oxidative stress, exhibits an intracellular localization and contributes to fungal virulence in the Galleria mellonella animal model system. FC levels were unaffected following deletion of all other genes encoding microsome-associated proteins. MirC does not appear to play a role in either siderophore export from, or uptake into, A. fumigatus. Label-free quantitative proteomic analysis unexpectedly revealed increased abundance of siderophore biosynthetic enzymes. In addition, increased expression of hapX (7.2 and 13.8-fold at 48 and 72 h, respectively; p < 0.001) was observed in ΔmirC compared to wild-type under iron-replete conditions by qRT-PCR. This was complemented by significantly elevated extracellular triacetylfusarinine C (TAFC; p < 0.01) and fusarinine C (FSC; p < 0.05) siderophore secretion. We conclude that MirC plays an important role in FC biosynthesis and contributes to the maintenance of iron homeostasis in A. fumigatus

Regulation of Nonribosomal Peptide Synthesis: bis-Thiomethylation Attenuates Gliotoxin Biosynthesis in Aspergillus fumigatus

Gliotoxin is a redox-active nonribosomal peptide produced by Aspergillus fumigatus. Like many other disulfide-containing epipolythiodioxopiperazines, a bis-thiomethylated form is also produced. In the case of gliotoxin, bisdethiobis(methylthio)gliotoxin (BmGT) is formed for unknown reasons by a cryptic enzyme. Here, we identify the S-adenosylmethionine- dependent gliotoxin bis-thiomethyltransferase (GtmA), which converts dithiogliotoxin to BmGT. This activity, which is induced by exogenous gliotoxin, is only detectable in protein lysates of A. fumigatus deficient in the gliotoxin oxidoreductase, gliT. Thus, GtmA is capable of substrate bis-thiomethylation. Deletion of gtmA completely abrogates BmGT formation and we now propose that the purpose of BmGT formation is primarily to attenuate gliotoxin biosynthesis. Phylogenetic analysis reveals 124 GtmA homologs within the Ascomycota phylum. GtmA is encoded outside the gliotoxin biosynthetic cluster and primarily serves to negatively regulate gliotoxin biosynthesis. This mechanism of postbiosynthetic regulation of nonribosomal peptide synthesis appears to be quite unusual

Comparative Genomic and Proteomic Analyses of Three Widespread Phytophthora Species: Phytophthora chlamydospora, Phytophthora gonapodyides and Phytophthora pseudosyringae

The genus includes some of the most devastating plant pathogens. Here we report draft genome sequences for three ubiquitous species-, , and . is an important forest pathogen that is abundant in Europe and North America. and are globally widespread species often associated with aquatic habitats. They are both regarded as opportunistic plant pathogens. The three sequenced genomes range in size from 45 Mb to 61 Mb. Similar to other oomycete species, tandem gene duplication appears to have played an important role in the expansion of effector arsenals. Comparative analysis of carbohydrate-active enzymes (CAZymes) across 44 oomycete genomes indicates that oomycete lifestyles may be linked to CAZyme repertoires. The mitochondrial genome sequence of each species was also determined, and their gene content and genome structure were compared. Using mass spectrometry, we characterised the extracellular proteome of each species and identified large numbers of proteins putatively involved in pathogenicity and osmotrophy. The mycelial proteome of each species was also characterised using mass spectrometry. In total, the expression of approximately 3000 genes per species was validated at the protein level. These genome resources will be valuable for future studies to understand the behaviour of these three widespread species

Comparative Genomic and Proteomic Analyses of Three Widespread Phytophthora Species: Phytophthora chlamydospora, Phytophthora gonapodyides and Phytophthora pseudosyringae

The genus includes some of the most devastating plant pathogens. Here we report draft genome sequences for three ubiquitous species-, , and . is an important forest pathogen that is abundant in Europe and North America. and are globally widespread species often associated with aquatic habitats. They are both regarded as opportunistic plant pathogens. The three sequenced genomes range in size from 45 Mb to 61 Mb. Similar to other oomycete species, tandem gene duplication appears to have played an important role in the expansion of effector arsenals. Comparative analysis of carbohydrate-active enzymes (CAZymes) across 44 oomycete genomes indicates that oomycete lifestyles may be linked to CAZyme repertoires. The mitochondrial genome sequence of each species was also determined, and their gene content and genome structure were compared. Using mass spectrometry, we characterised the extracellular proteome of each species and identified large numbers of proteins putatively involved in pathogenicity and osmotrophy. The mycelial proteome of each species was also characterised using mass spectrometry. In total, the expression of approximately 3000 genes per species was validated at the protein level. These genome resources will be valuable for future studies to understand the behaviour of these three widespread species

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P<0.001) and H2O2 (P<0.01), unexpectedly, exogenous gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P<0.05) the growth of the gliK26933 deletion mutant. The A. fumigatus gliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse- phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus gliK26933 mutant than in those of the wild type (P0.0024 [fold difference, 24] and P0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus gliK26933 mutant compared to the wild type (P<0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the gliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus