Multi-pathways-mediated mechanisms of selenite reduction and elemental selenium nanoparticles biogenesis in the yeast-like fungus Aureobasidium melanogenum I15.

Selenium (Se) plays a critical role in diverse biological processes and is widely used across manufacturing industries. However, the contamination of Se oxyanions also poses a major public health concern. Microbial transformation is a promising approach to detoxify Se oxyanions and produce elemental selenium nanoparticles (SeNPs) with versatile industrial potential. Yeast-like fungi are an important group of environmental microorganisms, but their mechanisms for Se oxyanions reduction remain unknown. In this study, we found that Aureobasidium melanogenum I15 can reduce 1.0 mM selenite by over 90% within 48 h and efficiently form intracellular or extracellular spherical SeNPs. Metabolomic and proteomic analyses disclosed that A. melanogenum I15 evolves a complicated selenite reduction mechanism involving multiple metabolic pathways, including the glutathione/glutathione reductase pathway, the thioredoxin/thioredoxin reductase pathway, the siderophore-mediated pathway, and multiple oxidoreductase-mediated pathways. This study provides the first report on the mechanism of selenite reduction and SeNPs biogenesis in yeast-like fungi and paves an alternative avenue for the bioremediation of selenite contamination and the production of functional organic selenium compounds.

The Development of Aspergillus flavus and Biosynthesis of Aflatoxin B1 are Regulated by the Golgi-Localized Mn2+ Transporter Pmr1.

Microorganisms rely on diverse ion transport and trace elements to sustain growth, development, and secondary metabolism. Manganese (Mn2+) is essential for various biological processes and plays a crucial role in the metabolism of human cells, plants, and yeast. In Aspergillus flavus, we confirmed that Pmr1 localized in cis- and medial-Golgi compartments was critical in facilitating Mn2+ transport, fungal growth, development, secondary metabolism, and glycosylation. In comparison to the wild type, the Δpmr1 mutant displayed heightened sensitivity to environmental stress, accompanied by inhibited synthesis of aflatoxin B1, kojic acid, and a substantial reduction in pathogenicity toward peanuts and maize. Interestingly, the addition of exogenous Mn2+ effectively rectified the developmental and secondary metabolic defects in the Δpmr1 mutant. However, Mn2+ supplement failed to restore the growth and development of the Δpmr1Δgdt1 double mutant, which indicated that the Gdt1 compensated for the functional deficiency of pmr1. In addition, our results showed that pmr1 knockout leads to an upregulation of O-glycosyl-N-acetylglucose (O-GlcNAc) and O-GlcNAc transferase (OGT), while Mn2+ supplementation can restore the glycosylation in A. flavus. Collectively, this study indicates that the pmr1 regulates Mn2+ via Golgi and maintains growth and metabolism functions of A. flavus through regulation of the glycosylation.

Valorization of spent coffee grounds through integrated bioprocess of fermentable sugars, volatile fatty acids, yeast-based single-cell protein and biofuels production.

Recovering nutrients from waste for biological processes aligns with sustainability principles. This study aimed to convert spent coffee grounds (SCG) into valuable products, including fermentable sugars, volatile fatty acids (VFAs), yeast-based single-cell protein and biofuels. Alkaline pretreatment was conducted before enzymatic hydrolysis, in which the pretreated SCG was hydrolyzed with varying enzyme loadings (20-60 filter paper units (FPU)/g-solid) and solid loadings (3-15 % w/v). The hydrolyzed slurry was utilized for VFAs and hydrogen production, yielding high values of 0.66 g/g-volatile solids (VS) and 109 mL/g-VS, respectively, using an enzyme loading of 50 FPU/g-solid and a solid loading of 3 % (w/v). The derived VFAs were used to cultivate a newly isolated yeast, Candida maltosa KKU-ARY2, resulting in an accumulated protein content of 43.7 % and a biomass concentration of 4.6 g/L. This study highlights the conversion of SCG into essential components, emphasizing the benefits of waste utilization through cascade bioprocesses.

Altered Expression of Two Small Secreted Proteins (ssp4 and ssp6) Affects the Degradation of a Natural Lignocellulosic Substrate by Pleurotus ostreatus.

Pleurotus ostreatus is a white-rot fungus that can degrade lignin in a preferential manner using a variety of extracellular enzymes, including manganese and versatile peroxidases (encoded by the vp1-3 and mnp1-6 genes, respectively). This fungus also secretes a family of structurally related small secreted proteins (SSPs) encoded by the ssp1-6 genes. Using RNA sequencing (RNA-seq), we determined that ssp4 and ssp6 are the predominant members of this gene family that were expressed by P. ostreatus during the first three weeks of growth on wheat straw. Downregulation of ssp4 in a strain harboring an ssp RNAi construct (KDssp1) was then confirmed, which, along with an increase in ssp6 transcript levels, coincided with reduced lignin degradation and the downregulation of vp2 and mnp1. In contrast, we observed an increase in the expression of genes related to pectin and side-chain hemicellulose degradation, which was accompanied by an increase in extracellular pectin-degrading capacity. Genome-wide comparisons between the KDssp1 and the wild-type strains demonstrated that ssp silencing conferred accumulated changes in gene expression at the advanced cultivation stages in an adaptive rather than an inductive mode of transcriptional response. Based on co-expression networking, crucial gene modules were identified and linked to the ssp knockdown genotype at different cultivation times. Based on these data, as well as previous studies, we propose that P. ostreatus SSPs have potential roles in modulating the lignocellulolytic and pectinolytic systems, as well as a variety of fundamental biological processes related to fungal growth and development.

Immunomodulatory Potential of Fungal Extracellular Vesicles: Insights for Therapeutic Applications.

Extracellular vesicles (EVs) are membranous vesicular organelles that perform a variety of biological functions including cell communication across different biological kingdoms. EVs of mammals and, to a lesser extent, bacteria have been deeply studied over the years, whereas investigations of fungal EVs are still in their infancy. Fungi, encompassing both yeast and filamentous forms, are increasingly recognized for their production of extracellular vesicles (EVs) containing a wealth of proteins, lipids, and nucleic acids. These EVs play pivotal roles in orchestrating fungal communities, bolstering pathogenicity, and mediating interactions with the environment. Fungal EVs have emerged as promising candidates for innovative applications, not only in the management of mycoses but also as carriers for therapeutic molecules. Yet, numerous questions persist regarding fungal EVs, including their mechanisms of generation, release, cargo regulation, and discharge. This comprehensive review delves into the present state of knowledge regarding fungal EVs and provides fresh insights into the most recent hypotheses on the mechanisms driving their immunomodulatory properties. Furthermore, we explore the considerable potential of fungal EVs in the realms of medicine and biotechnology. In the foreseeable future, engineered fungal cells may serve as vehicles for tailoring cargo- and antigen-specific EVs, positioning them as invaluable biotechnological tools for diverse medical applications, such as vaccines and drug delivery.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Discovery of Pyrazolone Carbothioamide Derivatives as Inhibitors of the Pdr1-KIX Interaction for Combinational Treatment of Azole-Resistant Candidiasis.

Candida glabrata has emerged as an important opportunistic pathogen of invasive candidiasis due to increasing drug resistance. Targeting Pdr1-KIX interactions with small molecules represents a potential strategy for treating drug-resistant candidiasis. However, effective Pdr1-KIX inhibitors are rather limited, hindering the validation of target druggability. Here, new Pdr1-KIX inhibitors were designed and assayed. Particularly, compound B8 possessed a new chemical scaffold and exhibited potent KIX binding affinity, leading to enhanced synergistic efficacy with fluconazole to treat resistant C. glabrata infection (FICI = 0.28). Compound B8 acted by inhibiting the efflux pump and down-regulating resistance-associated genes through blocking the Pdr1-KIX interaction. Compound B8 exhibited excellent in vitro and in vivo antifungal potency in combination with fluconazole against azole-resistant C. glabrata. It also had direct antifungal effect to treat C. glabrata infection, suggesting new mechanisms of action independent of Pdr1-KIX inhibition. Therefore, compound B8 represents a promising lead compound for antifungal drug development.

Biodiesel Production Using Palm Oil with a MOF-Lipase B Biocatalyst from Candida Antarctica: A Kinetic and Thermodynamic Study.

This research presents the results of the immobilization of Candida Antarctica Lipase B (CALB) on MOF-199 and ZIF-8 and its use in the production of biodiesel through the transesterification reaction using African Palm Oil (APO). The results show that the highest adsorption capacity, the 26.9 mg·g-1 Lipase, was achieved using ZIF-8 at 45 °C and an initial protein concentration of 1.20 mg·mL-1. The results obtained for the adsorption equilibrium studies allow us to infer that CALB was physically adsorbed on ZIF-8 while chemically adsorbed with MOF-199. It was determined that the adsorption between Lipase and the MOFs under study better fit the Sips isotherm model. The results of the kinetic studies show that adsorption kinetics follow the Elovich model for the two synthesized biocatalysts. This research shows that under the experimental conditions in which the studies were carried out, the adsorption processes are a function of the intraparticle and film diffusion models. According to the results, the prepared biocatalysts showed a high efficiency in the transesterification reaction to produce biodiesel, with methanol as a co-solvent medium. In this work, the catalytic studies for the imidazolate, ZIF-8, presented more catalytic activity when used with CALB. This system presented 95% biodiesel conversion, while the biocatalyst formed by MOF-199 and CALB generated a catalytic conversion percentage of 90%. Although both percentages are high, it should be noted that CALB-MOF-199 presented better reusability, which is due to chemical interactions.

Co-immunoprecipitation-Based Identification of Effector-Host Protein Interactions from Pathogen-Infected Plant Tissue.

Protein-protein interactions play an essential role in host-pathogen interactions. Phytopathogens secrete a cocktail of effector proteins to suppress plant immunity and reprogram host cell metabolism in their favor. Identification and characterization of effectors and their target protein complexes by co-immunoprecipitation can help to gain a deeper understanding of the functions of individual effectors during pathogenicity and can also provide new insights into the wiring of plant signaling pathways or metabolic complexes. Here we describe a detailed protocol to perform co-immunoprecipitation of effector-target protein complexes from plant extracts with an example of the Ustilago maydis/maize pathosystem for which we also provide a fungal protoplast transformation and maize seedling infection protocols.

The role of manganese in morphogenesis and pathogenesis of the opportunistic fungal pathogen Candida albicans.

Metals such as Fe, Cu, Zn, and Mn are essential trace nutrients for all kingdoms of life, including microbial pathogens and their hosts. During infection, the mammalian host attempts to starve invading microbes of these micronutrients through responses collectively known as nutritional immunity. Nutritional immunity for Zn, Fe and Cu has been well documented for fungal infections; however Mn handling at the host-fungal pathogen interface remains largely unexplored. This work establishes the foundation of fungal resistance against Mn associated nutritional immunity through the characterization of NRAMP divalent metal transporters in the opportunistic fungal pathogen, Candida albicans. Here, we identify C. albicans Smf12 and Smf13 as two NRAMP transporters required for cellular Mn accumulation. Single or combined smf12Δ/Δ and smf13Δ/Δ mutations result in a 10-80 fold reduction in cellular Mn with an additive effect of double mutations and no losses in cellular Cu, Fe or Zn. As a result of low cellular Mn, the mutants exhibit impaired activity of mitochondrial Mn-superoxide dismutase 2 (Sod2) and cytosolic Mn-Sod3 but no defects in cytosolic Cu/Zn-Sod1 activity. Mn is also required for activity of Golgi mannosyltransferases, and smf12Δ/Δ and smf13Δ/Δ mutants show a dramatic loss in cell surface phosphomannan and in glycosylation of proteins, including an intracellular acid phosphatase and a cell wall Cu-only Sod5 that is key for oxidative stress resistance. Importantly, smf12Δ/Δ and smf13Δ/Δ mutants are defective in formation of hyphal filaments, a deficiency rescuable by supplemental Mn. In a disseminated mouse model for candidiasis where kidney is the primary target tissue, we find a marked loss in total kidney Mn during fungal invasion, implying host restriction of Mn. In this model, smf12Δ/Δ and smf13Δ/Δ C. albicans mutants displayed a significant loss in virulence. These studies establish a role for Mn in Candida pathogenesis.

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification.

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.

Five Post-Translational Modification Residues of CmPT2 Play Key Roles in Yeast and Rice.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the largest cut flowers in the world. Phosphate transporter Pht1 family member CmPht1;2 protein (CmPT2) plays an important role in response to low-phosphate (LP) stress in chrysanthemum. Post-translational modification (PTM) can modulate the function of proteins in multiple ways. Here, we used yeast and rice systems to study the role of putative PTM in CmPT2 by determining the effect of mutation of key amino acid residues of putative glycosylation, phosphorylation, and myristoylation sites. We chose nine amino acid residues in the putative PTM sites and mutated them to alanine (A) (Cmphts). CmPT2 recovered the growth of yeast strain MB192 under LP conditions. However, G84A, G222A, T239A, Y242A, and N422A mutants could not grow normally under LP conditions. Analysis of phosphorus absorption kinetics showed that the Km of CmPT2 was 65.7 µM. Among the nine Cmphts, the expression of five with larger Km (124.4-397.5 µM) than CmPT2 was further evaluated in rice. Overexpression of CmPT2-OE increased plant height, effective panicle numbers, branch numbers, and yield compared with that of wild type 'Wuyunjing No. 7' (W7). Overexpression of Cmphts-OE led to decreased plant height and effective panicle numbers compared with that of the CmPT2-OE strain. The Pi content in roots of CmPT2-OE was higher than that of the W7 under both high (normal) phosphate (HP) and LP conditions. However, the Pi content in the leaves and roots was significantly lower in the N422A-OE strain than in the CmPT2-OE strain under both HP and LP conditions. Under LP conditions, the phosphorus starvation response (PSR) genes in CmPT2-OE were inhibited at the transcription level. The expression patterns of phosphorus-related genes in T239A, Y242A, and N422A-OE under LP conditions were different from those of CmPT2-OE. In conclusion, these five post-translational modification residues of CmPT2 play key roles in modulating the function of CmPT2. This work boosters our understanding of the function of phosphate transporters and provides genetic resources for improving the efficiency of phosphorus utilization in crop plants.

Filamentous fungi for sustainable vegan food production systems within a circular economy: Present status and future prospects.

Filamentous fungi serve as potential candidates in the production of different value-added products. In the context of food, there are several advantages of using filamentous fungi for food. Among the main advantages is that the fungal biomass used food not only meets basic nutritional requirements but that it is also rich in protein, low in fat, and free of cholesterol. This speaks to the potential of filamentous fungi in the production of food that can substitute animal-derived protein sources such as meat. Moreover, life-cycle analyses and techno-economic analyses reveal that fungal proteins perform better than animal-derived proteins in terms of land use efficiency as well as global warming. The present article provides an overview of the potential of filamentous fungi as a source of food and food supplements. The commercialization potential as well as social, legal and safety issues of fungi-based food products are discussed.

Study of Serial Exposures of an Astringent Green Tea Flavonoid Extract with Oral Cell-Based Models.

It is well known that repeated exposure to phenolic compounds (PCs) raises astringency perception. However, the link between this increase and the oral cavity's interactions with salivary proteins (SPs) and other oral constituents is unknown. To delve deeper into this connection, a flavonoid-rich green tea extract was tested in a series of exposures to two oral cell-based models using a tongue cell line (HSC3) and a buccal mucosa cell line (TR146). Serial exposures show cumulative PC binding to all oral models at all concentrations of the green tea extract; however, the contribution for the first and second exposures varies. The tongue mucosal pellicle (HSC3-Mu-SP) may contribute more to first-stage astringency (retaining 0.15 ± 0.01 mg mL-1 PCs at the first exposure), whereas the buccal mucosal pellicle (TR146-Mu-SP) retained significantly less (0.08 ± 0.02 mg mL-1). Additionally, increased salivary volume (SV+), which simulates the stimulation of salivary flow brought by a food stimulus, significantly enhances PC binding, particularly for TR146 cells: TR46-Mu-SP_SV+ bound significantly higher total PC concentration (0.17 ± 0.02 mg mL-1) than the model without increased salivary volume TR146-Mu-SP_SV- (0.09 ± 0.03 mg mL-1). This could be associated with a higher contribution of these oral cells for astringency perception during repeated exposures. Furthermore, PCs adsorbed in the first exposure to cell monolayer models (+TR146 and +HSC3) change the profile of PCs bound to these models in the second exposure. Regarding the structure binding activity, PCs with a total higher number of hydroxyl groups were more bound by the models containing SP. Regarding the SP, basic proline-rich proteins (bPRPs) may be involved in the increased perception of astringency upon repeated exposures. The extent of bPRP precipitation by PCs in mucosal pellicle models for both cell lines (HSC3 and TR146) in the second exposure (76 ± 13 and 83 ± 6%, respectively) was significantly higher than in the first one (25 ± 14 and 5 ± 6%, respectively).

Penicillium chrysogenum as a fungal factory for feruloyl esterases.

Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: • Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. • Overexpression of the ORFs produced a novel P. rubens strain with improved activity. • The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown.

Glutamine Synthetase Contributes to the Regulation of Growth, Conidiation, Sclerotia Development, and Resistance to Oxidative Stress in the Fungus Aspergillus flavus.

The basic biological function of glutamine synthetase (Gs) is to catalyze the conversion of ammonium and glutamate to glutamine. This synthetase also performs other biological functions. However, the roles of Gs in fungi, especially in filamentous fungi, are not fully understood. Here, we found that conditional disruption of glutamine synthetase (AflGsA) gene expression in Aspergillus flavus by using a xylose promoter leads to a complete glutamine deficiency. Supplementation of glutamine could restore the nutritional deficiency caused by AflGsA expression deficiency. Additionally, by using the xylose promoter for the downregulation of AflgsA expression, we found that AflGsA regulates spore and sclerotic development by regulating the transcriptional levels of sporulation genes abaA and brlA and the sclerotic generation genes nsdC and nsdD, respectively. In addition, AflGsA was found to maintain the balance of reactive oxygen species (ROS) and to aid in resisting oxidative stress. AflGsA is also involved in the regulation of light signals through the production of glutamine. The results also showed that the recombinant AflGsA had glutamine synthetase activity in vitro and required the assistance of metal ions. The inhibitor molecule L-α-aminoadipic acid suppressed the activity of rAflGsA in vitro and disrupted the morphogenesis of spores, sclerotia, and colonies in A. flavus. These results provide a mechanistic link between nutrition metabolism and glutamine synthetase in A. flavus and suggest a strategy for the prevention of fungal infection.

Structural basis for activation of fungal sterol receptor Upc2 and azole resistance.

Fungal transcription factor Upc2 senses ergosterol levels and regulates sterol biosynthesis and uptake. Constitutive activation of Upc2 causes azole resistance in Candida species. We determined the structure of ergosterol-bound Upc2, revealing the ligand specificity and transcriptional regulation. Ergosterol binding involves conformational changes of the ligand-binding domain, creating a shape-complementary hydrophobic pocket. The conserved helix α12 and glycine-rich loop are critical for sterol recognition by forming the pocket wall. The mutations of the glycine-rich loop inhibit ligand binding by steric clashes and constitutively activate Upc2. The translocation of Upc2 is regulated by Hsp90 chaperone in a sterol-dependent manner. Ergosterol-bound Upc2 associates with Hsp90 using the C-terminal tail, which retains the inactive Upc2 in the cytosol. Ergosterol dissociation induces a conformational change of the C-terminal tail, releasing Upc2 from Hsp90 for nuclear transport by importin α. The understanding of the regulatory mechanism provides an antifungal target for the treatment of azole-resistant Candida infections.

Stabilization of immobilized lipases by treatment with metallic phosphate salts.

Lipases from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida rugosa (CRL), forms A and B of lipase from Candida antarctica (CALA and CALB) and Eversa Transform 2.0 have been immobilized on octyl-agarose beads at two different loads (1 mg/g and saturated support) and treated with phosphate and/or some metallic salts (Zn2+, Co2+, Cu2+). They have been also immobilized on the support modified by the metallic phosphate, usually driving to biocatalyst with lower stability or marginal improvements. The effects of the phosphate/metal modification on enzyme features depended on the loading of the support. Some enzymes (TLL, CRL or CALA), mainly using the highly loaded biocatalysts, showed very significant improvement on enzyme stability after the treatment with some of the metal phosphates (next to a 20-fold factor), improvements that were not justified by the presence of metallic or phosphate ions in solution, as they had negative effects on enzyme stabilities. In some other cases, a significant increase in enzyme activity was detected (e.g., CALB). This could be explained by the modification of the nucleation places of the enzymes by the metallic phosphate, and this could help to explain the good results obtained in the nanoflower immobilization of many enzymes.

Physiological and transcriptional profiling of surfactin exerted antifungal effect against Candida albicans.

Given the risk of Candida albicans overgrowth in the gut, novel complementary therapies should be developed to reduce fungal dominancy. This study highlights the antifungal characteristics of a Bacillus subtilis-derived secondary metabolite, surfactin with high potential against C. albicans. Surfactin inhibited the growth of C. albicans following a 1-hour exposure, in addition to reduced adhesion and morphogenesis. Specifically, surfactin did not affect the level of reactive oxygen species but increased the level of reduced glutathione. Surprisingly, ethanol production was increased following 2 h of surfactin exposure. Surfactin treatment caused a significant reduction in intracellular iron, manganese and zinc content compared to control cells, whereas the level of copper was not affected. Alongside these physiological properties, surfactin also enhanced fluconazole efficacy. To gain detailed insights into the surfactin-related effects on C. albicans, genome-wide gene transcription analysis was performed. Surfactin treatment resulted in 1390 differentially expressed genes according to total transcriptome sequencing (RNA-Seq). Of these, 773 and 617 genes with at least a 1.5-fold increase or decrease in transcription, respectively, were selected for detailed investigation. Several genes involved in morphogenesis or related to metabolism (e.g., glycolysis, ethanol and fatty acid biosynthesis) were down-regulated. Moreover, surfactin decreased the expression of ERG1, ERG3, ERG9, ERG10 and ERG11 involved in ergosterol synthesis, whereas genes associated with ribosome biogenesis and iron metabolism and drug transport-related genes were up-regulated. Our data demonstrate that surfactin significantly influences the physiology and gene transcription of C. albicans, and could contribute to the development of a novel innovative complementary therapy.

Immunomodulatory protein from ganoderma microsporum protects against oxidative damages and cognitive impairments after traumatic brain injury.

A traumatic brain injury (TBI) causes abnormal proliferation of neuroglial cells, and over-release of glutamate induces oxidative stress and inflammation and leads to neuronal death, memory deficits, and even death if the condition is severe. There is currently no effective treatment for TBI. Recent interests have focused on the benefits of supplements or natural products like Ganoderma. Studies have indicated that immunomodulatory protein from Ganoderma microsporum (GMI) inhibits oxidative stress in lung cancer cells A549 and induces cancer cell death by causing intracellular autophagy. However, no evidence has shown the application of GMI on TBI. Thus, this study addressed whether GMI could be used to prevent or treat TBI through its anti-inflammation and antioxidative effects. We used glutamate-induced excitotoxicity as in vitro model and penetrating brain injury as in vivo model of TBI. We found that GMI inhibits the generation of intracellular reactive oxygen species and reduces neuronal death in cortical neurons against glutamate excitotoxicity. In neurite injury assay, GMI promotes neurite regeneration, the length of the regenerated neurite was even longer than that of the control group. The animal data show that GMI alleviates TBI-induced spatial memory deficits, expedites the restoration of the injured areas, induces the secretion of brain-derived neurotrophic factors, increases the superoxide dismutase 1 (SOD-1) and lowers the astroglial proliferation. It is the first paper to apply GMI to brain-injured diseases and confirms that GMI reduces oxidative stress caused by TBI and improves neurocognitive function. Moreover, the effects show that prevention is better than treatment. Thus, this study provides a potential treatment in naturopathy against TBI.