Coordination of two regulators SscA and VosA in Aspergillus nidulans conidia.

Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans.

A lipase from Lacticaseibacillus rhamnosus IDCC 3201 with thermostability and pH resistance for use as a detergent additive.

Lipases are important biocatalysts and ubiquitous in plants, animals, and microorganisms. The high growth rates of microorganisms with low production costs have enabled the wide application of microbial lipases in detergent, food, and cosmetic industries. Herein, a novel lipase from Lacticaseibacillus rhamnosus IDCC 3201 (Lac-Rh) was isolated and its activity analyzed under a range of reaction conditions to evaluate its potential industrial application. The isolated Lac-Rh showed a molecular weight of 24 kDa and a maximum activity of 3438.5 ± 1.8 U/mg protein at 60 °C and pH 8. Additionally, Lac-Rh retained activity in alkaline conditions and in 10% v/v concentrations of organic solvents, including glycerol and acetone. Interestingly, after pre-incubation in the presence of multiple commercial detergents, Lac-Rh maintained over 80% of its activity and the stains from cotton were successfully removed under a simulated laundry  setting. Overall, the purified lipase from L. rhamnosus IDCC 3201 has potential for use as a detergent in industrial applications. KEY POINTS: • A novel lipase (Lac-Rh) was isolated from Lacticaseibacillus rhamnosus IDCC 3201 • Purified Lac-Rh exhibited its highest activity at a temperature of 60 °C and a pH of 8, respectively • Lac-Rh remains stable in commercial laundry detergent and enhances washing performance.

Design Principles of Large Cation Incorporation in Halide Perovskites.

Perovskites have stood out as excellent photoactive materials with high efficiencies and stabilities, achieved via cation mixing techniques. Overcoming challenges to the stabilization of Perovskite solar cells calls for the development of design principles of large cation incorporation in halide perovskite to accelerate the discovery of optimal stable compositions. Large fluorinated organic cations incorporation is an attractive method for enhancing the intrinsic stability of halide perovskites due to their high dipole moment and moisture-resistant nature. However, a fluorinated cation has a larger ionic size than its non-fluorinated counterpart, falling within the upper boundary of the mixed-cation incorporation. Here, we report on the intrinsic stability of mixed Methylammonium (MA) lead halides at different concentrations of large cation incorporation, namely, ehtylammonium (EA; [CH3CH2NH3]+) and 2-fluoroethylammonium (FEA; [CH2FCH2NH3]+). Density functional theory (DFT) calculations of the enthalpy of the mixing and analysis of the perovskite structural features enable us to narrow down the compositional search domain for EA and FEA cations around concentrations that preserve the perovskite structure while pointing towards the maximal stability. This work paves the way to developing design principles of a large cation mixture guided by data analysis of DFT data. Finally, we present the automated search of the minimum enthalpy of mixing by implementing Bayesian optimization over the compositional search domain. We introduce and validate an automated workflow designed to accelerate the compositional search, enabling researchers to cut down the computational expense and bias to search for optimal compositions.

The role of the VosA-repressed dnjA gene in development and metabolism in Aspergillus species.

The DnaJ family of proteins (or J-proteins) are molecular chaperones that govern protein folding, degradation, and translocation in many organisms. Although J-proteins play key roles in eukaryotic and prokaryotic biology, the role of J-proteins in Aspergillus species is currently unknown. In this study, we characterized the dnjA gene, which encodes a putative DnaJ protein, in two Aspergillus species: Aspergillus nidulans and Aspergillus flavus. Expression of the dnjA gene is inhibited by the velvet regulator VosA, which plays a pivotal role in spore survival and metabolism in Aspergillus. The deletion of dnjA decreased the number of asexual spores (conidia), produced abnormal conidiophores, and reduced sexual fruiting bodies (cleistothecia) or sclerotia. In addition, the absence of dnjA caused increased sterigmatocystin or aflatoxin production in A. nidulans and A. flavus, respectively. These results suggest that DnjA plays a conserved role in asexual and sexual development and mycotoxin production in Aspergillus species. However, DnjA also plays a species-specific role; AniDnjA but not AflDnjA, affects conidial viability, trehalose contents, and thermal tolerance of conidia. In plant virulence assay, the infection ability of the ΔAfldnjA mutant decreased in the kernels, suggesting that DnjA plays a crucial role in the pathogenicity of A. flavus. Taken together, these results demonstrate that DnjA is multifunctional in Aspergillus species; it is involved in diverse biological processes, including fungal differentiation and secondary metabolism.

Correction: Exploring new approaches towards the formability of mixed-ion perovskites by DFT and machine learning.

Correction for 'Exploring new approaches towards the formability of mixed-ion perovskites by DFT and machine learning' by Heesoo Park et al., Phys. Chem. Chem. Phys., 2019, DOI: 10.1039/c8cp06528d.

Exploring new approaches towards the formability of mixed-ion perovskites by DFT and machine learning.

Recent years have witnessed a growing effort in engineering and tuning the properties of hybrid halide perovskites as light absorbers. These have led to the successful enhancement of their stability, a feature that is often counterbalanced by a reduction of their power-conversion efficiency. In order to provide a systematic analysis of the structure-property relationships of this class of compounds we have performed density functional theory calculations exploring fully inorganic ABC3 chalcogenide (I-V-VI3), halide (I-II-VII3) and hybrid perovskites. Special attention has been given to structures featuring three-dimensional BC6 octahedral networks because of their efficient carrier transport properties. In particular we have carefully analyzed the role of BC6 octahedral deformations, rotations and tilts in the thermodynamic stability and optical properties of the compounds. By using machine learning algorithms we have estimated the relations between the octahedral deformation and the bandgap, and established a similarity map among all the calculated compounds.

Learn-and-Match Molecular Cations for Perovskites.

Forecasting the structural stability of hybrid organic/inorganic compounds, where polyatomic molecules replace atoms, is a challenging task; the composition space is vast, and the reference structure for the organic molecules is ambiguously defined. In this work, we use a range of machine-learning algorithms, constructed from state-of-the-art density functional theory data, to conduct a systematic analysis on the likelihood of a given cation to be housed in the perovskite structure. In particular, we consider both ABC3 chalcogenide (I-V-VI3) and halide (I-II-VII3) perovskites. We find that the effective atomic radius and the number of lone pairs residing on the A-site cation are sufficient features to describe the perovskite phase stability. Thus, the presented machine-learning approach provides an efficient way to map the phase stability of the vast class of compounds, including situations where a cation mixture replaces a single A-site cation. This work demonstrates that advanced electronic structure theory combined with machine-learning analysis can provide an efficient strategy superior to the conventional trial-and-error approach in materials design.

Calcineurin Targets Involved in Stress Survival and Fungal Virulence.

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

Elucidating the Impact of Chalcogen Content on the Photovoltaic Properties of Oxychalcogenide Perovkskites: NaMO3-x Qx (M=Nb, Ta; Q=S, Se, Te).

In the quest for nontoxic and stable perovskites for solar cells, we have conducted a systematic study of the effect of chalcogen content in oxychalcogenide perovskite by using DFT and quasi-particle perturbation theory. We explored the changes in the electronic structure due to the substitution of O atoms in NaNbO3 and NaTaO3 perovskite structures with various chalcogens (S, Se, Te) at different concentrations. Interestingly, the introduction of the chalcogen atoms resulted in a drastic reduction in the electronic band gap, which made some of the compounds fall within the visible range of the solar spectrum. In addition, our analysis of the electronic structure shows that the optical transition becomes direct as a result of the strong hybridization between the orbitals of the transition metal and those of the chalcogen ion, in contrast to the indirect band feature of NaNbO3 and NaTaO3 . We identified candidates with a high theoretical solar conversion efficiency that approached the Shockley-Queisser limit, which makes them suitable for thin-film solar cell applications. The present work serves as a guideline for experimental efforts by identifying the chalcogen content that should be targeted during the synthetic route of thermodynamically stable and strongly photoactive absorbers for oxychalcogenide perovskites in thin-film solar cells.

Improved Photoactivity of Pyroxene Silicates by Cation Substitutions.

We investigated the possibility of band structure engineering of pyroxene silicates with chemical formula A+1 B+3 Si2 O6 by proper cation substitution. Typically, band gaps of naturally formed pyroxene silicates such as NaAlSi2 O6 are quite high (≈5 eV). Therefore, it is important to find a way to reduce band gaps for these materials below 3 eV to make them usable for optoelectronic applications operating at visible light range of the spectrum. Using first-principles calculations, we found that appropriate substitutions of both A+ and B3+ cations can reduce the band gaps of these materials to as low as 1.31 eV. We also discuss how the band gap in this class of materials is affected by cation radii, electronegativity of constituent elements, spin-orbit coupling, and structural modifications. In particular, the replacement of Al3+ in NaAlSi2 O6 by another trivalent cation Tl3+ results in the largest band-gap reduction and emergence of intermediate bands. We also found that all considered materials are still thermodynamically stable. This work provides a design approach for new environmentally benign and abundant materials for use in photovoltaics and optoelectronic devices.

High molecular weight genomic DNA mini-prep for filamentous fungi.

Purification of high quality genomic DNA (gDNA) from filamentous fungi suitable for whole genome sequencing has previously involved many steps. Here, we report a simple and easy-to-follow mini-preparation protocol for high molecular weight (∼20kb) gDNA from filamentous fungi including Aspergillus and Eurotium. This comprehensive protocol includes graphic step-by-step instructions for inoculation, homogenization, and purification of gDNA. The most critical step is a thorough 3-5min homogenization of the freeze-dried mycelium using a motorized hand-held homogenizer with a mini spatula inserted. Approximately 20mg of the fine mycelial powder is then subjected to a modified procedure for the DNeasy Plant Mini Kit (Qiagen). This Qiagen spin column protocol avoids precipitation, dryness, and resuspension of gDNA, which can cause shearing and loss of gDNA. Final gDNA yields from ∼20mg of fine mycelial powder are 8 to 20µg with a consistent 260/280nm absorbance ratio of ∼1.9. All 30 gDNA samples we purified using our method were of high molecular weight (∼20kb). Whole genome sequencing of these DNA samples resulted in 160-260 X coverage with 2×150 reads using NextSeq 500. These gDNAs are also of a suitable quality for Southern blotting and PCR-based amplification of various genes in filamentous fungi.

Diversity, Application, and Synthetic Biology of Industrially Important Aspergillus Fungi.

The filamentous fungal genus Aspergillus consists of over 340 officially recognized species. A handful of these Aspergillus fungi are predominantly used for food fermentation and large-scale production of enzymes, organic acids, and bioactive compounds. These industrially important Aspergilli primarily belong to the two major Aspergillus sections, Nigri and Flavi. Aspergillus oryzae (section Flavi) is the most commonly used mold for the fermentation of soybeans, rice, grains, and potatoes. Aspergillus niger (section Nigri) is used in the industrial production of various enzymes and organic acids, including 99% (1.4 million tons per year) of citric acid produced worldwide. Better understanding of the genomes and the signaling mechanisms of key Aspergillus species can help identify novel approaches to enhance these commercially significant strains. This review summarizes the diversity, current applications, key products, and synthetic biology of Aspergillus fungi commonly used in industry.

In Vitro Activities of LCB 01-0648, a Novel Oxazolidinone, against Gram-Positive Bacteria.

Oxazolidinones are a novel class of synthetic antibacterial agents that inhibit bacterial protein synthesis. Here, we synthesized and tested a series of oxazolidinone compounds containing cyclic amidrazone. Among these compounds, we further investigated the antibacterial activities of LCB01-0648 against drug-susceptible or resistant Gram-positive cocci in comparison with those of six reference compounds. LCB01-0648 showed the most potent antimicrobial activities against clinically isolated Gram-positive bacteria. Against the methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCNS) isolates, LCB01-0648 showed the lowest MIC90s (0.5 mg/L) among the tested compounds. In addition, LCB01-0648 had the lowest minimum inhibitory concentrations (MICs) against the four linezolid-resistant S. aureus (LRSA) strains (range 2-4 mg/L). The results of the time-kill studies demonstrated that LCB01-0648 at a concentration 8× the (MIC) showed bactericidal activity against methicillin-susceptible Staphylococcus aureus MSSA or MRSA, but showed a bacteriostatic effect against LRSA. These results indicate that LCB01-0648 could be a good antibacterial candidate against multidrug-resistant (MDR) Gram-positive cocci.

In Vivo Activity of LCB 01-0699, a Prodrug of LCB 01-0648, against Staphylococcus aureus.

LCB01-0648 is a novel oxazolidinone compound that shows potent antibacterial activities against most Gram-positive cocci, including the multi-drug resistant Staphylococcusaureus. In this study, in vivo activity of LCB01-0699, a LCB01-0648 prodrug, against S.aureus was evaluated in comparison with that of Linezolid. The results of the systemic infection study demonstrated that LCB01-0699 was more potent than Linezolid against methicillin-susceptible and -resistant S. aureus strains. The in vivo efficacy of LCB01-0699 against methicillin-susceptible and -resistant S. aureus strains in a skin infection model showed more potent activity than Linezolid. LCB01-0699 shows potent in vivo activity against methicillin-susceptible and -resistant S. aureus strains, suggesting that LCB01-0699 would be a novel candidate for the treatment of these infectious diseases caused by S. aureus.

The velvet family of fungal regulators contains a DNA-binding domain structurally similar to NF-κB.

Morphological development of fungi and their combined production of secondary metabolites are both acting in defence and protection. These processes are mainly coordinated by velvet regulators, which contain a yet functionally and structurally uncharacterized velvet domain. Here we demonstrate that the velvet domain of VosA is a novel DNA-binding motif that specifically recognizes an 11-nucleotide consensus sequence consisting of two motifs in the promoters of key developmental regulatory genes. The crystal structure analysis of the VosA velvet domain revealed an unforeseen structural similarity with the Rel homology domain (RHD) of the mammalian transcription factor NF-κB. Based on this structural similarity several conserved amino acid residues present in all velvet domains have been identified and shown to be essential for the DNA binding ability of VosA. The velvet domain is also involved in dimer formation as seen in the solved crystal structures of the VosA homodimer and the VosA-VelB heterodimer. These findings suggest that defence mechanisms of both fungi and animals might be governed by structurally related DNA-binding transcription factors.

Antimicrobial Activity of Zabofloxacin against Clinically Isolated Streptococcus pneumoniae.

Zabofloxacin is a novel fluoroquinolone agent that has potent activity against gram-positive pathogens. In this study, we confirmed that zabofloxacin showed the most potent in vitro and in vivo activities against drug-resistant Streptococcus pneumoniae. Among the fluoroquinolone compounds, zabofloxacin showed the most potent in vitro activity against clinical isolates of penicillin-sensitive S. pneumoniae (minimum inhibitory concentration, MIC90: 0.03 mg/L) and penicillin-resistant S. pneumoniae (MIC90: 0.03 mg/L). Against quinolone-resistant S. pneumoniae, zabofloxacin (MIC90: 1 mg/L) was more active than ciprofloxacin, sparfloxacin, and moxifloxacin; however, its activity was the same as that of gemifloxacin. The in vivo activity of zabofloxacin was most potent among the quinolone compounds tested against the systemic infection and respiratory tract infection models in mice.

Core oxidative stress response in Aspergillus nidulans.

BACKGROUND: The b-Zip transcription factor AtfA plays a key role in regulating stress responses in the filamentous fungus Aspergillus nidulans. To identify the core regulons of AtfA, we examined genome-wide expression changes caused by various stresses in the presence/absence of AtfA using A. nidulans microarrays. We also intended to address the intriguing question regarding the existence of core environmental stress response in this important model eukaryote. RESULTS: Examination of the genome wide expression changes caused by five different oxidative stress conditions in wild type and the atfA null mutant has identified a significant number of stereotypically regulated genes (Core Oxidative Stress Response genes). The deletion of atfA increased the oxidative stress sensitivity of A. nidulans and affected mRNA accumulation of several genes under both unstressed and stressed conditions. The numbers of genes under the AtfA control appear to be specific to a stress-type. We also found that both oxidative and salt stresses induced expression of some secondary metabolite gene clusters and the deletion of atfA enhanced the stress responsiveness of additional clusters. Moreover, certain clusters were down-regulated by the stresses tested. CONCLUSION: Our data suggest that the observed co-regulations were most likely consequences of the overlapping physiological effects of the stressors and not of the existence of a general environmental stress response. The function of AtfA in governing various stress responses is much smaller than anticipated and/or other regulators may play a redundant or overlapping role with AtfA. Both stress inducible and stress repressive regulations of secondary metabolism seem to be frequent features in A. nidulans.

SscA is required for fungal development, aflatoxin production, and pathogenicity in Aspergillus flavus.

Fungal spores are specialized dormant cells that act as primary reproductive biological particles and exhibit strong viability under extremely harsh conditions. They contaminate a variety of crops and foods, causing severe health hazards to humans and animals. Previous studies demonstrated that a spore-specific transcription factor SscA plays pivotal roles in the conidiogenesis of the model organism Aspergillus nidulans. In this study, we investigated the biological and genetic functions of SscA in the aflatoxin-producing fungus A. flavus. Deletion of sscA showed reduced conidia formation, lost long-term viability, and exhibited more sensitivity to thermal, oxidative, and radiative stresses. The sscA-deficient strain showed increased aflatoxin B1 production in conidia as well as mycelia. Importantly, the absence of sscA affected fungal pathogenicity on crops. Further transcriptomic and phenotypic studies suggested that SscA coordinates conidial wall structures. Overall, SscA is important for conidial formation, maturation and dormancy, mycotoxin production, and pathogenicity in A. flavus.

The MYB-like protein MylA contributes to conidiogenesis and conidial germination in Aspergillus nidulans.

Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA expression of genes. In this study, we identified and characterized one MYB-like protein in the model organism Aspergillus nidulans. We screened the mRNA levels of genes encoding MYB-like proteins containing two MYB repeats in conidia and found that the mRNA levels of four genes including flbD, cicD, and two uncharacterized genes, were high in conidia. To investigate the roles of two uncharacterized genes, AN4618 and AN10944, deletion mutants for each gene were generated. Our results revealed that AN4618 was required for fungal development. Therefore, we further investigated the role of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats. Functional studies revealed that MylA was essential for normal fungal growth and development. Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia. In addition, the germination rate of the mylA deletion mutant conidia was decreased compared with that of the wild-type conidia. Overall, this study suggests that MylA is critical for appropriate development, conidial maturation, dormancy, and germination in A. nidulans.

Replica exchange molecular dynamics for Li-intercalation in graphite: a new solution for an old problem.

Li intercalation and graphite stacking have been extensively studied because of the importance of graphite in commercial Li-ion batteries. Despite this attention, there are still questions about the atomistic structures of the intermediate states that exist during lithiation, especially when phase dynamics cause a disordered Li distribution. The Li migration event (diffusion coefficient of 10-5 nm2 ns-1) makes it difficult to explore the various Li-intercalation configurations in conventional molecular dynamics (MD) simulations with an affordable simulation timescale. To overcome these limitations, we conducted a comprehensive study using replica-exchange molecular dynamics (REMD) in combination with the ReaxFF force field. This approach allowed us to study the behavior of Li-intercalated graphite from any starting arrangement of Li at any value of x in LixC6. Our focus was on analyzing the energetic favorability differences between the relaxed structures. We rationalized the trends in formation energy on the basis of observed structural features, identifying three main structural features that cooperatively control Li rearrangement in graphite: Li distribution, graphite stacking mode and gallery height (graphene layer spacing). We also observed a tendency for clustering of Li, which could lead to dynamic local structures that approximate the staging models used to explain intercalation into graphite.