Gliotoxin-mediated bacterial growth inhibition is caused by specific metal ion depletion.

Overcoming antimicrobial resistance represents a formidable challenge and investigating bacterial growth inhibition by fungal metabolites may yield new strategies. Although the fungal non-ribosomal peptide gliotoxin (GT) is known to exhibit antibacterial activity, the mechanism(s) of action are unknown, although reduced gliotoxin (dithiol gliotoxin; DTG) is a zinc chelator. Furthermore, it has been demonstrated that GT synergises with vancomycin to inhibit growth of Staphylococcus aureus. Here we demonstrate, without precedent, that GT-mediated growth inhibition of both Gram positive and negative bacterial species is reversed by Zn2+ or Cu2+ addition. Both GT, and the known zinc chelator TPEN, mediate growth inhibition of Enterococcus faecalis which is reversed by zinc addition. Moreover, zinc also reverses the synergistic growth inhibition of E. faecalis observed in the presence of both GT and vancomycin (4 µg/ml). As well as zinc chelation, DTG also appears to chelate Cu2+, but not Mn2+ using a 4-(2-pyridylazo)resorcinol assay system and Zn2+ as a positive control. DTG also specifically reacts in Fe3+-containing Siderotec™ assays, most likely by Fe3+ chelation from test reagents. GSH or DTT show no activity in these assays. Confirmatory high resolution mass spectrometry, in negative ion mode, confirmed, for the first time, the presence of both Cu[DTG] and Fe[DTG]2 chelates. Label free quantitative proteomic analysis further revealed major intracellular proteomic remodelling within E. faecalis in response to GT exposure for 30-180 min. Globally, 4.2-7.2% of detectable proteins exhibited evidence of either unique presence/increased abundance or unique absence/decreased abundance (n = 994-1160 total proteins detected), which is the first demonstration that GT affects the bacterial proteome in general, and E. faecalis, specifically. Unique detection of components of the AdcABC and AdcA-II zinc uptake systems was observed, along with apparent ribosomal reprofiling to zinc-free paralogs in the presence of GT. Overall, we hypothesise that GT-mediated bacterial growth inhibition appears to involve intracellular zinc depletion or reduced bioavailability, and based on in vitro chelate formation, may also involve dysregulation of Cu2+ homeostasis.

Immunoproteomic analysis of the serum IgG response to cell wall-associated proteins of Staphylococcus aureus strains belonging to CC97 and CC151.

CC97 and CC151 are two of the most common Staphylococcus aureus lineages associated with bovine intramammary infection. The genotype of the infecting S. aureus strain influences virulence and the progression of intramammary disease. Strains from CC97 and CC151 encode a distinct array of virulence factors. Identification of proteins elaborated in vivo will provide insights into the molecular mechanism of pathogenesis of these lineages, as well as facilitating the development of tailored treatments and pan-lineage vaccines and diagnostics. The repertoire of genes encoding cell wall-anchored (CWA) proteins was identified for S. aureus strains MOK023 (CC97) and MOK124 (CC151); MOK023 encoded more CWA proteins than MOK124. Serum collected during an in vivo challenge trial was used to investigate whether the humoral response to cell wall proteins was strain-specific. Immunoproteomic analysis demonstrated that the humoral response in MOK023-infected cows predominantly targeted high molecular weight proteins while the response in MOK124-infected cows targeted medium or low molecular weight proteins. Antigenic proteins were identified by two-dimensional serum blotting followed by mass spectometry-based identification of immunoreactive spots, with putative antigens subsequently validated. The CWA proteins ClfB, SdrE/Bbp and IsdA were identified as immunogenic regardless of the infecting strain. In addition, a number of putative strain-specific imunogens were identified. The variation in antigens produced by different strains may indicate that these strains have different strategies for exploiting the intramammary niche. Such variation should be considered when developing novel control strategies including vaccines, therapeutics and diagnostics.

Gliotoxin and related metabolites as zinc chelators: implications and exploitation to overcome antimicrobial resistance.

Antimicrobial resistance (AMR) is a major global problem and threat to humanity. The search for new antibiotics is directed towards targeting of novel microbial systems and enzymes, as well as augmenting the activity of pre-existing antimicrobials. Sulphur-containing metabolites (e.g., auranofin and bacterial dithiolopyrrolones [e.g., holomycin]) and Zn2+-chelating ionophores (PBT2) have emerged as important antimicrobial classes. The sulphur-containing, non-ribosomal peptide gliotoxin, biosynthesised by Aspergillus fumigatus and other fungi exhibits potent antimicrobial activity, especially in the dithiol form (dithiol gliotoxin; DTG). Specifically, it has been revealed that deletion of the enzymes gliotoxin oxidoreductase GliT, bis-thiomethyltransferase GtmA or the transporter GliA dramatically sensitise A. fumigatus to gliotoxin presence. Indeed, the double deletion strain A. fumigatus ΔgliTΔgtmA is especially sensitive to gliotoxin-mediated growth inhibition, which can be reversed by Zn2+ presence. Moreover, DTG is a Zn2+ chelator which can eject zinc from enzymes and inhibit activity. Although multiple studies have demonstrated the potent antibacterial effect of gliotoxin, no mechanistic details are available. Interestingly, reduced holomycin can inhibit metallo-ß-lactamases. Since holomycin and gliotoxin can chelate Zn2+, resulting in metalloenzyme inhibition, we propose that this metal-chelating characteristic of these metabolites requires immediate investigation to identify new antibacterial drug targets or to augment the activity of existing antimicrobials. Given that (i) gliotoxin has been shown in vitro to significantly enhance vancomycin activity against Staphylococcus aureus, and (ii) that it has been independently proposed as an ideal probe to dissect the central 'Integrator' role of Zn2+ in bacteria - we contend such studies are immediately undertaken to help address AMR.

Eating Disorders: Identification and Management in General Medical and Psychiatric Settings.

OBJECTIVE: Eating disorders (EDs) are serious, complex illnesses with both behavioral and physical health features. EDs have high rates of medical and psychiatric morbidity, and a 6% mortality rate, the highest of any mental illness. Early detection of EDs offers the best opportunity for recovery; yet, estimates are that as few as one in 10 individuals with an ED receive treatment. The purpose of this article is to provide an ED identification and management overview for inpatient nurse clinicians in general psychiatric and medical settings, helping to facilitate timely recognition and care. METHOD: An overview of ED diagnostic criteria and two evidence-based ED tools are introduced for consideration. RESULTS: Opportunities to identify and help manage an ED are numerous. Most individuals with an ED make several health care visits in either medical or psychiatric settings without ever being screened for an ED. General ED screening and assessment tool familiarization can facilitate a treatment trajectory for these patients, improve overall quality of life, and may potentially result in a life-saving intervention for this often-deadly cluster of medical and psychiatric disorders. CONCLUSION: Screening and assessment in general clinical settings, identifying patients with undiagnosed EDs, beginning basic treatment plans, and referrals for appropriate follow-up care, have the potential to reduce ED recidivism and related health care costs. Simultaneously, and most important, long-term outcomes for patients with EDs may improve.

HMO-primed bifidobacteria exhibit enhanced ability to adhere to intestinal epithelial cells.

The ability of gut commensals to adhere to the intestinal epithelium can play a key role in influencing the composition of the gut microbiota. Bifidobacteria are associated with a multitude of health benefits and are one of the most widely used probiotics for humans. Enhanced bifidobacterial adhesion may increase host-microbe, microbe-nutrient, and/or microbe-microbe interactions, thereby enabling consolidated health benefits to the host. The objective of this study was to determine the ability of human milk oligosaccharides (HMOs) to enhance bifidobacterial intestinal adhesion in vitro. This study assessed the colonisation-promoting effects of HMOs on four commercial infant-associated Bifidobacterium strains (two B. longum subsp. infantis strains, B. breve and B. bifidum). HT29-MTX cells were used as an in vitro intestinal model for bacterial adhesion. Short-term exposure of four commercial infant-associated Bifidobacterium strains to HMOs derived from breastmilk substantially increased the adherence (up to 47%) of these probiotic strains. Interestingly, when strains were incubated with HMOs as a four-strain combination, the number of viable bacteria adhering to intestinal cells increased by >90%. Proteomic analysis of this multi-strain bifidobacterial mixture revealed that the increased adherence resulting from exposure to HMOs was associated with notable increases in the abundance of sortase-dependent pili and glycosyl hydrolases matched to Bifidobacterium bifidum. This study suggests that HMOs may prime infant gut-associated Bifidobacterium for colonisation to intestinal epithelial cells by influencing the expression of various colonization factors.

Quantitative Proteomic Analysis Reveals Yeast Cell Wall Products Influence the Serum Proteome Composition of Broiler Chickens.

With an ever-growing market and continual financial pressures associated with the prohibition of antibiotic growth promoters, the poultry industry has had to rapidly develop non-antibiotic alternatives to increase production yields. A possible alternative is yeast and its derivatives, such as the yeast cell wall (YCW), which have been proposed to confer selected beneficial effects on the host animal. Here, the effect of YCW supplementation on the broiler chicken was investigated using a quantitative proteomic strategy, whereby serum was obtained from three groups of broilers fed with distinct YCW-based Gut Health Products (GHP) or a control basal diet. Development of a novel reagent enabled application of ProteoMiner™ technology for sample preparation and subsequent comparative quantitative proteomic analysis revealed proteins which showed a significant change in abundance (n = 167 individual proteins; p < 0.05); as well as proteins which were uniquely identified (n = 52) in, or absent (n = 37) from, GHP-fed treatment groups versus controls. An average of 7.1% of proteins showed changes in abundance with GHP supplementation. Several effects of these GHPs including immunostimulation (via elevated complement protein detection), potential alterations in the oxidative status of the animal (e.g., glutathione peroxidase and catalase), stimulation of metabolic processes (e.g., differential abundance of glyceraldehyde-3-phosphate dehydrogenase), as well as evidence of a possible hepatoprotective effect (attenuated levels of serum α-glutathione s-transferase) by one GHP feed supplement, were observed. It is proposed that specific protein detection may be indicative of GHP efficacy to stimulate broiler immune status, i.e., may be biomarkers of GHP efficacy. In summary, this work has developed a novel technology for the preparation of high dynamic range proteomic samples for LC-MS/MS analysis, is part of the growing area of livestock proteomics and, importantly, provides evidential support for beneficial effects that GHP supplementation has on the broiler chicken.

Immunoproteomic analysis of the secretome of bovine-adapted strains of Staphylococcus aureus demonstrates a strain-specific humoral response.

Staphylococcus aureus is a common pathogen associated with bovine intramammary infection. A number of distinct S. aureus lineages are associated with such infections although there is a dearth of knowledge regarding the major immunogenic antigens associated with each lineage and whether these antigens provide protection against heterologous strains. Identification of the major immunogenic antigens of the predominant bovine-adapted S. aureus lineages would assist in the design of effective vaccines and diagnostic tests to control intramammary infections caused by S. aureus. The aim of this study was to characterise the serum IgG response to S. aureus extracellular proteins in cows infected with strains from different lineages, as well as to identify antigenic proteins produced by these strains. Genotypic characterisation found that strain MOK124 (CC151) encoded more toxins, including the ruminant-specific leukocidin LukMF, compared to strain MOK023 (CC97). In addition, MOK124 secreted more toxins in vitro, compared to MOK023. Immunoproteomic analysis was performed using sera from cows infected with either MOK023 or MOK124. One-dimensional serum blotting revealed that cows infected with MOK023 predominantly generated a humoral response against high molecular weight proteins while cows infected with MOK124 primarily generated a humoral response against low molecular weight proteins. Two-dimensional serum blotting demonstrated that antibodies produced by an MOK023 infected cow could cross react with some of the extracellular proteins produced by MOK124 and vice versa. Mass spectrometry analysis of immunoreactive proteins identified common candidate immunogens produced by both strains, including α-hemolysin and ß-hemolysin. In addition, strain-specific candidate immunogens were also identified. This study demonstrates that genes encoding important S. aureus secreted virulence factors, the production of cognate gene products, and the humoral immune response to infection is, to an extent, strain-dependent. However, the identification of some common candidate immunogens suggests that there are proteins that can be exploited for further vaccine or diagnostic research that targets S. aureus strains from a variety of lineages.

Comparative Structural and Compositional Analyses of Cow, Buffalo, Goat and Sheep Cream.

Factors affecting milk and milk fraction composition, such as cream, are poorly understood, with most research and human health application associated with cow cream. In this study, proteomic and lipidomic analyses were performed on cow, goat, sheep and Bubalus bubalis (from now on referred to as buffalo), bulk milk cream samples. Confocal laser scanning microscopy was used to determine the composition, including protein, lipid and their glycoconjugates, and the structure of the milk fat globules. BLAST2GO was used to annotate functional indicators of cream protein. Functional annotation of protein highlighted a broad level of similarity between species. However, investigation of specific biological process terms revealed distinct differences in antigen processing and presentation, activation, and production of molecular mediators of the immune response. Lipid analyses revealed that saturated fatty acids were lowest in sheep cream and similar in the cream of the other species. Palmitic acid was highest in cow and lowest in sheep cream. Cow and sheep milk fat globules were associated with thick patches of protein on the surface, while buffalo and goat milk fat globules were associated with larger areas of aggregated protein and significant surface adsorbed protein, respectively. This study highlights the differences between cow, goat, sheep, and buffalo milk cream, which can be used to support their potential application in functional foods such as infant milk formula.

At the metal-metabolite interface in Aspergillus fumigatus: towards untangling the intersecting roles of zinc and gliotoxin.

Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc-depleted medium, addition of zinc (1 µM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, triacetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 µM zinc) and ΔgtmA (at >1 µM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 µM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidoreductase- and secondary metabolism-related enzymes was also evident in a zinc- and strain-dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatus.

Saúde mental, transtorno por uso de substâncias e transtorno por uso de opioides: atualizações e estratégias de tratamento / Mental health, substance use disorder, and opioid uses disorder: updates and strategies for treatment / Salud mental, trastorno por uso de sustancias y trastorno por uso de opioides: actualizaciones y estrategias de tratamiento

OBJETIVO: descrever as principais estratégias para abordar lacunas na identificação, tratamento e treinamento sobre saúde mental, transtorno do uso de substâncias (TUS) e transtorno do uso de opioides (TUO). MÉTODO: trata-se de uma revisão narrativa, a partir de artigos recentes e de publicações de instituições que abordam a temática da saúde mental e da dependência química reconhecidas internacionalmente. RESULTADOS: a prevalência de uso concomitante de substâncias e transtornos psiquiátricos/de saúde mental tem sido elevada e continua crescente, compondo problemas complexos que implicam em desafios de tratamento multifacetados, incluindo condições médicas, deficiências, falta de moradia, abandono de medicamentos e altas taxas de recaída. O tratamento de TUS's e TUO's são questões individualmente complexas. A combinação dos dois transtornos requer uma abordagem de diagnóstico e tratamento dedicada e multifacetada. CONCLUSÃO: como a prevalência de TUO's, TUS's e COD's continua a aumentar, enfermeiros e profissionais de saúde devem estar preparados para diagnosticar, tratar e/ou encaminhar os usuários para garantir o cuidado adequado e a recuperação a longo prazo dos indivíduos acometidos.

OBJECTIVE: to describe the main strategies to deal with gaps in the identification, treatment and training regarding substance use disorder (SUD), and opioid uses disorder (OUD). METHOD: this is a narrative review, based on recent articles and publications on mental health and substance use recognized internationally. RESULTS: a prevalence of co-occurring substance use and mental health/psychiatric disorders continue to rise and are considered complex problems, with multifaceted treatment challenges including medical conditions, disabilities, homelessness, medication noncompliance, and high relapse rates. The treatment for SUD and OUD are complex. The co-occurrence of these two disorders require a multifaceted approach for the diagnosis and treatment. CONCLUSION: the prevalence of SUD, OUD and their co-occurrence continue to rise and nurses and other health professionals should be prepared to diagnose, treat and/or refer users to assure their adequate care and long term recovery.

OBJETIVO: describir las principales estrategias para abordar las brechas en la identificación, tratamiento y capacitación en salud mental, trastorno por uso de sustancias (TUS) y trastorno por uso de opioides (TUO). MÉTODO: se trata de una revisión narrativa, basada en artículos y publicaciones recientes de instituciones reconocidas internacionalmente que abordan el tema de la salud mental y la dependencia química. RESULTADOS: la prevalencia del uso concomitante de sustancias y trastornos psiquiátricos/de salud mental ha sido alta y continúa creciente, lo que agrava problemas complejos que implican desafíos de tratamiento multifacéticos, que incluyen afecciones médicas, discapacidades, falta de vivienda, abandono del uso de medicaciones y elevadas tasas de recaída. El tratamiento de los TUS y TUO son problemas individualmente complejo. Una combinación de los dos requiere un enfoque de diagnóstico y tratamiento dedicado y multifacético. CONCLUSIÓN: como la prevalencia de TUO, TUS y COD sigue aumentando, las enfermeras y los profesionales de la salud deben estar preparados para diagnosticar, tratar y / o encaminar para garantizar la atención adecuada y la recuperación a largo plazo de las personas afectadas.

Proteomic profiling and glycomic analysis of the yeast cell wall in strains with Aflatoxin B1 elimination ability.

The use of microorganisms for Aflatoxin B1 elimination has been studied as a new alternative tool and it is known that cell wall carried out a critical role. For that reason, cell wall and soluble intracellular fraction of eight yeasts with AFB1 detoxification capability were analysed. The quantitative and qualitative comparative label-free proteomic allowed the identification of diverse common constituent proteins, which revealed that putative cell wall proteins entailed less than 10% of the total proteome. It was possible to characterize different enzymes linked to cell wall polysaccharides biosynthesis as well as other proteins related with the cell wall organization and regulation. Additionally, the concentration of the principal polysaccharides was determined which permitted us to observe that ß-glucans concentration was higher than mannans in most of the samples. In order to better understand the biosorption role of the cell wall against the AFB1 , an antimycotic (Caspofungin) was used to damage the cell wall structure. This assay allowed the observation of an effect on the normal growth of those yeasts with damaged cell walls that were exposed to AFB1 . This effect was not observed in yeast with intact cell walls, which may reveal a protective role of this structure against mycotoxins.

Effects of antifungal agents on the fungal proteome: informing on mechanisms of sensitivity and resistance.

INTRODUCTION: Antifungal agents are essential in the fight against serious fungal disease, however emerging resistance is threatening an already limited collection of therapeutics. Proteomic analyses of effects of antifungal agents can expand our understanding of multifactorial mechanisms of action and have also proven valuable to elucidate proteomic changes associated with antifungal resistance. AREAS COVERED: This review covers the application of proteomic techniques to examine sensitivity and resistance to antifungals including commonly used therapeutics, amphotericin B, echinocandins and the azoles, based predominantly on studies involving Aspergillus fumigatus, Candida albicans and Candida glabrata from the last 10 years. In addition, non-clinical antimicrobial agents are also discussed, which highlight the potential of proteomics to identify new antifungal targets. EXPERT COMMENTARY: Fungal proteomics has evolved in the last decade with increased genome availability and developments in mass spectrometry. Collectively, these have led to the advancement of proteomic techniques, allowing increased coverage of the proteome. Gel-based proteomics laid the foundation for these types of studies, which has now shifted to the more powerful gel-free proteomics. This has resulted in the identification of key mediators and potential biomarkers of antifungal resistance, as well as elucidating the mechanisms of action of novel and established antifungal agents.

Hallmarks of Basidiomycete Soft- and White-Rot in Wood-Decay -Omics Data of Two Armillaria Species.

Wood-decaying Basidiomycetes are among the most efficient degraders of plant cell walls, making them key players in forest ecosystems, global carbon cycle, and in bio-based industries. Recent insights from -omics data revealed a high functional diversity of wood-decay strategies, especially among the traditional white-rot and brown-rot dichotomy. We examined the mechanistic bases of wood-decay in the conifer-specialists Armillaria ostoyae and Armillaria cepistipes using transcriptomic and proteomic approaches. Armillaria spp. (Fungi, Basidiomycota) include devastating pathogens of temperate forests and saprotrophs that decay wood. They have been discussed as white-rot species, though their response to wood deviates from typical white-rotters. While we observed an upregulation of a diverse suite of plant cell wall degrading enzymes, unlike white-rotters, they possess and express an atypical wood-decay repertoire in which pectinases and expansins are enriched, whereas lignin-decaying enzymes (LDEs) are generally downregulated. This combination of wood decay genes resembles the soft-rot of Ascomycota and appears widespread among Basidiomycota that produce a superficial white rot-like decay. These observations are consistent with ancestral soft-rot decay machinery conserved across asco- and Basidiomycota, a gain of efficient lignin-degrading ability in white-rot fungi and repeated, complete, or partial losses of LDE encoding gene repertoires in brown- and secondarily soft-rot fungi.

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence.

There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. Here, we have performed a comprehensive characterization and validation of the promising target methionine synthase (MetH). We show that in Aspergillus fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. Interestingly, growth can be recovered in the presence of certain metabolites, which shows that metH is a conditionally essential gene and consequently should be targeted in established infections for a more comprehensive validation. Accordingly, we have validated the use of the tetOFF genetic model in fungal research and improved its performance in vivo to achieve initial validation of targets in models of established infection. We show that repression of metH in growing hyphae halts growth in vitro, which translates into a beneficial effect when targeting established infections using this model in vivo Finally, a structure-based virtual screening of methionine synthases reveals key differences between the human and fungal structures and unravels features in the fungal enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is a valuable target for the development of new antifungals.IMPORTANCE Fungal pathogens are responsible for millions of life-threatening infections on an annual basis worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance has emerged and already become a serious threat to our capacity to treat fungal diseases. The first step to develop new drugs is often to identify molecular targets in the pathogen whose inhibition during infection can prevent its growth. However, the current models are not suitable to validate targets in established infections. Here, we have characterized the promising antifungal target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus fumigatus as a model. We have uncovered the underlying reason for its essentiality and confirmed its druggability. Furthermore, we have optimized the use of a genetic system to show a beneficial effect of targeting methionine synthase in established infections. Therefore, we believe that antifungal drugs to target methionine synthase should be pursued and additionally, we provide a model that permits gaining information about the validity of antifungal targets in established infections.

Differential distribution and proteomic response of Saccharomyces cerevisiae and non-model yeast species to zinc.

Zinc surplus in yeast cells has been previously investigated thanks to transcriptomic studies by using traditionally Saccharomyces cerevisiae as a model. However, proteome response under zinc-replete conditions needs to be further studied in yeast. For that reason, eight yeast strains from seven different species were inoculated in zinc-depleted and zinc-replete media. The quantitative and qualitative comparative label-free proteomic analysis enabled the identification of between 2000 and 3000 proteins from each strain, and changes to the proteome ranged from 2.5% to 43.7% of identified proteins. Functional analysis (Blast2Go) has allowed the characterization of differentially abundant proteins. Common zinc-responsive proteins have been detected for the eight strains such as oxidoreductases and transferases (increased in abundance) although more of the changes detected were not shared by all the strains tested. Zinc distribution under replete conditions has been analysed in cell wall fractions, and cytoplasm plus organelles (intracellular fraction), with the latter identified to be the main zinc reservoir. Additionally, the energy dispersive spectroscopy coupled to the scanning electron microscopy technique has permitted the visualization of zinc in the whole cell. Proteomic analysis revealed that while there were some shared responses, the non-model yeast species also showed distinct proteomic profiles in zinc-replete conditions, compared to S. cerevisiae, revealing new zinc-responsive proteins in yeast.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Proteomic investigation of interhyphal interactions between strains of Agaricus bisporus.

Hyphae of filamentous fungi undergo polar extension, bifurcation and hyphal fusion to form reticulating networks of mycelia. Hyphal fusion or anastomosis, a ubiquitous process among filamentous fungi, is a vital strategy for how fungi expand over their substrate and interact with or recognise self- and non-self hyphae of neighbouring mycelia in their environment. Morphological and genetic characterisation of anastomosis has been studied in many model fungal species, but little is known of the direct proteomic response of two interacting fungal isolates. Agaricus bisporus, the most widely cultivated edible mushroom crop worldwide, was used as an in vitro model to profile the proteomes of interacting cultures. The globally cultivated strain (A15) was paired with two distinct strains; a commercial hybrid strain and a wild isolate strain. Each co-culture presented a different interaction ranging from complete vegetative compatibility (self), lack of interactions, and antagonistic interactions. These incompatible strains are the focus of research into disease-resistance in commercial crops as the spread of intracellular pathogens, namely mycoviruses, is limited by the lack of interhyphal anastomosis. Unique proteomic responses were detected between all co-cultures. An array of cell wall modifying enzymes, plus fungal growth and morphogenesis proteins were found in significantly (P < 0.05) altered abundances. Nitrogen metabolism dominated in the intracellular proteome, with evidence of nitrogen starvation between competing, non-compatible cultures. Changes in key enzymes of A. bisporus morphogenesis were observed, particularly via increased abundance of glucanosyltransferase in competing interactions and certain chitinases in vegetative compatible interactions only. Carbohydrate-active enzyme arsenals are expanded in antagonistic interactions in A. bisporus. Pathways involved in carbohydrate metabolism and genetic information processing were higher in interacting cultures, most notably during self-recognition. New insights into the differential response of interacting strains of A. bisporus will enhance our understanding of potential barriers to viral transmission through vegetative incompatibility. Our results suggest that a differential proteomic response occurs between A. bisporus at strain-level and findings from this work may guide future proteomic investigation of fungal anastomosis.

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

The Phytophthora genus includes some of the most devastating plant pathogens. Here we report draft genome sequences for three ubiquitous Phytophthora species-Phytophthora chlamydospora, Phytophthora gonapodyides, and Phytophthora pseudosyringae. Phytophthora pseudosyringae is an important forest pathogen that is abundant in Europe and North America. Phytophthora chlamydospora and Ph. gonapodyides 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 Phytophthora species.

An Exploratory Study of a 3-Minute Mindfulness Intervention on Compassion Fatigue in Nurses.

This study shows that breathing mindfully for 3 minutes over a period of 4 weeks, positively affects compassion fatigue in nurses. A nonrandomized, pre/postintervention study was conducted using a 3-minute attentional breathing intervention. Thirty-two nurses participated over 4 weeks. The intervention demonstrated statistically significant reductions in compassion fatigue measures.