Cage-modified hypocrellin against multidrug-resistant Candida spp. with unprecedented activity in light-triggered combinational photodynamic therapy.

Infections caused by multidrug-resistant fungi pose a devastating threat to human health worldwide, making new antifungal strategies urgently desired. Antimicrobial photodynamic therapy (aPDT) has gained increasing attention due to its potential in fighting against fungal infection. However, the preparation of highly efficient and water-soluble photosensitizers (PSs) for this purpose remains a challenge. Herein, we present a new strategy to prepare powerful PSs for efficient aPDT by introducing a porous cage compound, which could facilitate the transportation of O2 and reactive oxygen species (ROS). Specifically, the natural PS hypocrellin A (HA) was attached to a novel organic cage compound (covalent organic polyhedra 1 tied, COP1T) with polyethylene glycol (PEG) chains to improve its water solubility. It was found that the resulting COP1T-HA exhibited in vitro antifungal efficiency several folds higher compared to the free HA in fighting against four types of multidrug-resistant fungal planktonic cells and biofilms, including the "super fungus" Candida auris. Interestingly, the red-shift of COP1T-HA adsorption led to the realization of phototheranostic aPDT for cage-modified HA or derivatives. Additionally, COP1T-HA exhibited good biocompatibility, excellent disinfection capacity and wound healing efficiency without obvious toxic effects in vivo of rat model. With further development and optimization, COP1T-HA has great potential to become a new class of antifungal agent to fight against drug-resistant pathogens.

Simultaneous partial nitrification, denitrification, and phosphorus removal in sequencing batch reactors via controlled reduced aeration and short-term sludge retention time decrease.

Simultaneous bio-treatment processes of organic carbon (C)-, nitrogen (N)-, and phosphorus (P)-containing wastewater are challenged by insufficient carbon sources in the effluent. In the present study, two parallel anaerobic/aerobic sequencing batch reactors (R-1 and R-2) treating low C/N (≤4) wastewater were employed using different partial nitrification start-up strategies, controlled reduced aeration, and decreased sludge retention time. Advanced removal efficiencies for NH4+-N (≥96%), total nitrogen (TN, ≥86%), PO43--P (≥95%), and CODintra (≥91%) were realized, with TN and PO43--P effluent concentrations of 10.0 ± 3.5 and 0.11 ± 0.3 mg/L in R-1 and 9.28 ± 4.0 and 0.11 ± 0.1 mg/L in R-2, respectively. Higher nitrite accumulation rate (nearly 100%) and TN (121.1 ± 0.7 mg TN/g VSS·d) and P (12.5 ± 0.6 mg PO43--P/g VSS·d) removal loadings were obtained in R-2 by a thorough elimination of nitrite-oxidizing bacteria. Moreover, different microbial structures and nutrient removal pathways were identified. Denitrifying glycogen-accumulating organisms (Candidatus Competibacter) and phosphorus-accumulating organisms (PAOs) (Tetrasphaera) removed N and P with partial nitrification-endogenous denitrification pathways and aerobic P removal in R-1. In R-2, aerobic denitrifying bacteria (Psychrobacter) and PAOs ensured N and P removal through the partial nitrification-aerobic denitrification and aerobic P removal pathways. Compared to R-1, R-2 offers greater efficiency, convenience, and scope to further reduce carbon-source demand.

Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology.

A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii). Plasmids containing single guide RNA and the methanol expression regulator 1 (MXR1) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single-base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris. The expression levels of several of the Mxr1-targeted genes, AOX1, AOX2, DAS1, and DAS2, in strains containing the various mutated variants of MXR1, were then detected through reverse transcription PCR following induction in methanol-containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1, DAS1, and DAS2, while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1, AOX2, DAS1, and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1, DAS1, and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.

Global identification of alternative splicing in Shiraia bambusicola and analysis of its regulation in hypocrellin biosynthesis.

Hypocrellins, as natural pigments from Shiraia bambusicola, have extensive applications in the agricultural, cosmetic, food, and feed industries, and play a vital role in photodynamic therapy for anticancer and antiviral treatments. However, environmental stresses are always the bottlenecks for increasing hypocrellin yield during the process of fermentation. Pre-mRNA alternative splicing (AS) is an essential mechanism in the defense of abiotic stresses in the animal and plant kingdom, but is seldom involved in fungi. In this study, AS from genome-wide sequencing and RNA-seq data for S. bambusicola was analyzed for the first time. Interestingly, the proportion of AS in S. bambusicola was 38.44% (most of them participated in metabolic processes, covering pigmentation and response to stimulus), a much higher ratio than seen in that of other fungal species (1.3-18%). Here, we identified the relationship of AS and secondary metabolic (SM) biosynthesis under a series of abiotic stresses. Suitable fungicides suppressed hypocrellin production significantly, and AS occurred in key functional genes (sbFLO, sbMFS, sbPKS) of hypocrellin biosynthesis. In contrast, H2O2 improved the yield of hypocrellins, but AS were not found in the corresponding gene cluster. A further study showed that overexpressing an isoform of sbPKS (sbPKSa) in Shiraia bambusicola could dramatically down-regulate the expression of the original gene sbPKS and nearly inhibit the production of hypocrellins. Altogether, our study strongly supported the hypothesis that AS had a vital role in the regulation of hypocrellin biosynthesis under stresses, and initially explored whether SM functional genes were relevant for fungi.

Antioxidant and antimicrobial activities of various extracts from Engleromyces sinensis fruiting body.

Engleromyces sinensis, as rare macro-ascomycetes and traditional ethnomedicine in the southeast part of China, have been applied in anti-infection, anti-inflammatory and anti-tumor for a long time. In this study, the antioxidant activities of ethyl acetate crude extract (EACE), acetone crude extract (ACE), 95% ethanol crude extract (ECE), methanol crude extract (MCE) and water crude extract (WCE) from E. sinensis fruiting body were investigated using conventional antioxidant assays in vitro for the first time. As results, it was noteworthy that WCE showed the greatest 2,2-diphenyl-1-picrylhydrazil (DPPH) radicals-scavenging activity and reducing power, with EC50 values of 3.56 and 19.28mg/mL. MCE and EACE exhibited higher hydroxyl radicals-scavenging activity and ferrous ion-chelating activity significantly, with EC50 values of 2.16 and 0.47mg/mL. The total phenolics and total polysaccharides content results revealed that WCE had the highest phenolics and polysaccharides contents with 1.19 mg GAEs/g extracts and 40.07 mg D-glucose/g extracts. The antimicrobial activity of the WCE, ECE, ACE, EACE was assessed in final and two of them, ACE and EACE showed a strong ability to inhibit the microbial growth. The research work demonstrated that E. sinensis fruiting body can present a promising source of antioxidant and antimicrobial agents.

Diversity and antimicrobial activity of culturable fungi from fishscale bamboo (Phyllostachys heteroclada) in China.

An important and useful bamboo species, fishscale bamboo (Phyllostachys heteroclada Oliver), is broadly distributed in Southeast China and has multiple purposes, including uses in cuisine, weaving, Chinese medicine and ecological protection. However, no previous studies have focused on the endophytes of this plant. In our article, a total of 127 fungal strains were first isolated from the healthy branches and leaves of common P. heteroclada. These endophytic fungi could be directly categorized into 50 morphotypes according to their culture characteristics, and their internal transcribed spacer (ITS) regions were analyzed for molecular identification. Using the BLAST search tool of the NCBI database and phylogenetic tree analysis, these isolates were divided into two phyla, Ascomycota (95.28%) and Basidiomycota (4.72%), including at least six orders (Xylariales, Capnodiales, Pleosporales, Hypocreales, Chaetothyriales and Polyporales) and fourteen genera (Arthrinium, Pestalotiopsis, Epicoccum, Cladosporium, Nigrospora, Setophoma, Didymella, Calcarisporium, Preussia, Nemania, Creosphaeria, Ophiobolus, Phialophora and Perenniporia). It is fascinating that four genera, Calcarisporium, Preussia, Creosphaeria and Phialophora were isolated from bamboos for the first time. The inhibitory effects against clinical pathogens were also preliminarily screened, and four isolates FB43 (Calcarisporium arbuscula), FB06 (Preussia minima), FB16 (Setophoma sp.) and FB21 (Perenniporia medulla-pains) among the candidate strains displayed broad-spectrum activities according to the agar diffusion method and the disk diffusion assay. Strain FB16 (Setophoma sp.) especially indicated high bioactivity against both clinical bacteria and yeast. This study is the first report on the diversity and antimicrobial activity of the endophytic fungi associated with P. heteroclada, which could be regarded as a potential source of drug precursors and could be used in biocontrol development.

High-efficiency biosynthesis of hypocrellin A in Shiraia sp. using gamma-ray mutagenesis.

Hypocrellin A (HA), well known as one of the best natural pigments and bioactive agent to treat skin diseases, is further anticipated to play a vital role in photodynamic therapy (PDT) in anticancer and antiviral treatments. In this study, an HA-producing strain ZZZ816 (Shiraia sp.) was isolated from the moso bamboo (Phyllostachys edulis) seeds, and gamma irradiation was used to mutagenize spores of the original strain. After treatment with cobalt-60 gamma ((60)Coγ) with different doses (20, 50, 80, 100, 150, 180, 300, and 500 Gy), the 100 Gy was selected as the optimal condition, which led to 77.2 % lethality of spores and 35 % positive mutant frequency. The extracted compound of the most excellent HA-producing strain (H-4-2) was precisely analyzed by a combination of seven detection methods, and the maximum HA content was shown to reach 2018.3 mg/L. HA production in H-4-2 increased by 414.9 % compared to that of original strain ZZZ816 (392 mg/L) and was significantly higher than all the other industrial HA-producing strains in published reports.

Identification and Evaluation of Reliable Reference Genes in the Medicinal Fungus Shiraia bambusicola.

The stability of reference genes plays a vital role in real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, which is generally regarded as a convenient and sensitive tool for the analysis of gene expression. A well-known medicinal fungus, Shiraia bambusicola, has great potential in the pharmaceutical, agricultural and food industries, but its suitable reference genes have not yet been determined. In the present study, 11 candidate reference genes in S. bambusicola were first evaluated and validated comprehensively. To identify the suitable reference genes for qRT-PCR analysis, three software-based algorithms, geNorm, NormFinder and Best Keeper, were applied to rank the tested genes. RNA samples were collected from seven fermentation stages using different media (potato dextrose or Czapek medium) and under different light conditions (12-h light/12-h dark and all-dark). The three most appropriate reference genes, ubi, tfc and ags, were able to normalize the qRT-PCR results under the culturing conditions of 12-h light/12-h dark, whereas the other three genes, vac, gke and acyl, performed better in the culturing conditions of all-dark growth. Therefore, under different light conditions, at least two reference genes (ubi and vac) could be employed to assure the reliability of qRT-PCR results. For both the natural culture medium (the most appropriate genes of this group: ubi, tfc and ags) and the chemically defined synthetic medium (the most stable genes of this group: tfc, vac and ef), the tfc gene remained the best gene used for normalizing the gene expression found with qRT-PCR. It is anticipated that these results would improve the selection of suitable reference genes for qRT-PCR assays and lay the foundation for an accurate analysis of gene expression in S. bambusicola.

Identification and characterization of microRNAs at different flowering developmental stages in moso bamboo (Phyllostachys edulis) by high-throughput sequencing.

Researching moso bamboo flowering has been difficult because of its unknown flowering interval and the rarity of florescent samples. To identify microRNAs (miRNAs) and study their expression patterns during the flower developmental process of moso bamboo, small RNAs from non-flowering leaves and four flower developmental periods were sequenced using Illumina technology. In total, 409 known miRNAs and 492 differentially expressed novel miRNAs were identified in moso bamboo. Of the known miRNAs that were differentially expressed between non-flowering and flowering samples, 64 were predicted to have a total of 308 targets. Among the miRNAs, seven known and five novel miRNAs were selected, as were four of their target genes, and their expression profiles were validated using qRT-PCR. The results indicated that the miRNA expression levels were negatively correlated with those of their targets. The research comprehensively revealed that the differentially expressed miRNAs and their targets participated in diverse biological pathways and played significant regulatory roles in moso bamboo flowering. The data provide a significant resource for understanding the molecular mechanisms in moso bamboo flowering and senescence, and serve as the primary foundation for further studies on metabolic regulatory networks that involve miRNAs.

Multigene phylogenetic analysis detects cryptic species of Tryblidiopsis in China.

Tryblidiopsis pinastri (Rhytismatales) has been recognized as being distributed throughout northern temperate regions. In the present study, comparative studies showed that species of Tryblidiopsis from China were distinct from the European type species. They are shown to belong to two new Tryblidiopsis species, T sichuanensis and T. sinensis, with a third known to be present in China but not described because the reference material is immature. Combined LSU rDNA, ITS rDNA and mtSSU rDNA sequences analyses revealed that Chinese species and European species formed a monophyletic clade. Collections of Tryblidiopsis from North America need to be re-examined and sequenced; they are probably not conspecific with T. pinastri. Tryblidiopsis species appear to be highly host specific and are probably restricted to the host genus Picea.

Municipal pipeline networks as preferential vapor intrusion pathways: A review.

Recent reports show a rise in instances where municipal networks, such as sewer lines, serve as pathways for vapor intrusion (VI), enabling volatile organic compounds (VOCs) vapors to travel along these networks. These VOCs pose potential health risks to occupants of buildings connected to these networks. Currently, there's a lack of specific technical or regulatory guidance on identifying and assessing the VI risk associated with sewer as preferential VI pathways. This critical review summarizes key findings from studies and site investigations related to sewer VI pathways. These findings cover background VOCs concentration levels in sewers, updates to site conceptual models, advances in sewer sampling techniques, innovative tools for identifying and characterizing sewer VI, and practices for assessing and mitigating sewer VI risk. While significant improvements have been made towards understanding how municipal pipeline networks act as VI pathways, more research is still needed to develop strategies for investigating sites and assessing risks associated with "pipeline VI pathways". Future research could focus on the development of "pipeline VI pathways" data set, the improvement and validation of investigation tools, and improving the understanding of VOCs transportation mechanisms within these "pipeline VI pathways".

A Phylogenetic and Taxonomic Revision of Discula theae-sinensis, the Causal Agents of Anthracnose on Camellia sinensis.

Tea (Camellia sinensis (L.) Kuntze) is one of the most important economic plants in China, and has many benefits for human health. Anthracnose is one of the most serious diseases of tea in China, and control of the fungus is important since most Chinese cultivars are susceptible to it. The agent of tea anthracnose was initially described as Gloeosporium theae-sinensis I. Miyake in Japan, which was later transferred to Discula, but this taxonomic position remains problematic. To shed light on these taxonomic and phylogenetic issues, the tea anthracnose pathogens were re-studied. Combining the morphological characteristics and a multigene phylogenetic analysis of nrITS, nrLSU, rpb2, and tef1 sequence data, a new genus Sinodiscula was proposed to accommodate the causal fungi of tea anthracnose, including a new species Sinodiscula camellicola and a new combination Sinodiscula theae-sinensis. Furthermore, the pathogenicity of the pathogens was determined according to Koch's postulates. This study thoroughly resolves the long-standing taxonomic and phylogenetic problems of the tea anthracnose pathogens.

Bacteriophage defends murine gut from Escherichia coli invasion via mucosal adherence.

Bacteriophage are sophisticated cellular parasites that can not only parasitize bacteria but are increasingly recognized for their direct interactions with mammalian hosts. Phage adherence to mucus is known to mediate enhanced antimicrobial effects in vitro. However, little is known about the therapeutic efficacy of mucus-adherent phages in vivo. Here, using a combination of in vitro gastrointestinal cell lines, a gut-on-a-chip microfluidic model, and an in vivo murine gut model, we demonstrated that a E. coli phage, øPNJ-6, provided enhanced gastrointestinal persistence and antimicrobial effects. øPNJ-6 bound fucose residues, of the gut secreted glycoprotein MUC2, through domain 1 of its Hoc protein, which led to increased intestinal mucus production that was suggestive of a positive feedback loop mediated by the mucus-adherent phage. These findings extend the Bacteriophage Adherence to Mucus model into phage therapy, demonstrating that øPNJ-6 displays enhanced persistence within the murine gut, leading to targeted depletion of intestinal pathogenic bacteria.

Optimisation of hypocrellin production in Shiraia-like fungi via genetic modification involving a transcription factor gene and a putative monooxygenase gene.

Shiraia-like fungi, which are rare parasitic fungi found around bamboo, play an important role in traditional medicine. Their main active component, hypocrellin, is widely used in medicine, food, and cosmetics. By comparing strains with different hypocrellin yields, we identified a transcription factor (SbTF) in the hypocrellin biosynthesis pathway. SbTF from high-yielding zzz816 and low-yielding CNUCC C72 differed in its protein structure. Subsequently, SbTF from high-yielding zzz816 was overexpressed in several strains. This stabilised the yield in zzz816 and significantly increased the yield in low-yielding CNUCC C72. Comparing downstream non-essential genes between wild type and SbTF-overexpressing CNUCC C72 showed that SbMNF was significantly up-regulated. Therefore, it was selected for further study. SbMNF overexpression increased the hypocrellin yield in low-yielding CNUCC C72 and altered the composition of compounds in high-yielding CNUCC 1353PR and zzz816. This involved an increased elsinochrome C yield in CNUCC 1353PR and an increased hypocrellin B yield in zzz816 (by 2 and 70.3 times that in the corresponding wild type, respectively). This study is the first to alter hypocrellin synthesis to alter the levels of one bioactive agent compared to another. The results provide new insights regarding genetic modification and will help to optimise fungal fermentation.

Four new species of Phyllosticta from China based on morphological and phylogenetic characterization.

Phyllosticta (Phyllostictaceae, Botryosphaeriales) species are widely distributed globally and constitute a diverse group of pathogenic and endophytic fungi associated with a broad range of plant hosts. In this study, four new species of Phyllosticta, i.e. P. endophytica, P. jiangxiensis, P. machili, and P. xinyuensis, were described using morphological characteristics and multi-locus phylogeny based on the internal transcribed spacer region (ITS) with intervening 5.8S rRNA gene, large subunit of rRNA gene (nrLSU), translation elongation factor 1-alpha gene (tef1), actin gene (act), and glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). Phyllosticta machili is the first species of this genus reported to infect plants of the Machilus genus.

Diversity and taxonomy of the genus Amanita (Amanitaceae, Agaricales) in the Yanshan Mountains, Northern China.

Globally, the species of Amanita are key components of ectomycorrhizal ecosystems. Some of them are widely known as poisonous or edible fungi. Although many new Amanita species from China have been described, the species diversity of Yanshan Mountains remains unknown. We here describe three new species, namely, A. borealis sp. nov. (Sect. Amanita), A. brunneola sp. nov. (Sect. Caesareae), and A. yanshanensis sp. nov. (Sect. Validae), based on morphological observations and molecular phylogenetic analyses. In addition, nine known species, namely, A. caesareoides (Sect. Caesareae), A. chiui (Sect. Vaginatae), A. muscaria (Sect. Amanita), A. oberwinklerana (Sect. Roanokenses), A. ovalispora (Sect. Vaginatae), A. subglobosa (Sect. Amanita), A. subjunquillea (Sect. phalloideae), A. vaginata var. vaginata (Sect. Vaginatae), and A. virosa (Sect. phalloideae), were reported from Yanshan Mountains for the first time. Our results emphasize that China has a high diversity of Amanita species and that additional studies are required to understand the exact species number. These findings play a crucial role in Amanita toxin research and ecological conservation. This study investigated the areas where Amanita species-related research is lacking. The study also attempted to better understand Amanita distribution and thus contribute to related research. This study enriches the species diversity of Amanita in Yanshan Mountains and offers additional data supporting the macrofungal systematics, toxin research, and diversity and ecological studies of Amanita in future studies.

A new species of Melanospora on truffles from China.

The new species Melanospora subterranea is described from China, based on morphological and phylogenetic analyses. This is the first record of Melanospora species parasitizing Chinese black and white truffles (Tuber indicum and T. huidongense), and its host range indicates that the new species might be a disease threat to commercially exploited European truffles including Tuber melanosporum and Tuber magnatum. Apparent polyphyly within the Ceratostomataceae can be explained at least in part by inadvertent sequencing of the host fungus instead of the parasite.

A new study of Nagrajomyces: with two new species proposed and taxonomic status inferred by phylogenetic methods.

Nagrajomyces (incertae sedis, Ascomycota) is a monotypic genus with a previously unknown systematic position. In this report, two new species are proposed, Nagrajomycesfusiformis and Nagrajomyceslaojunshanensis. These new taxa are proposed based on morphological characteristics evident via light microscopy and molecular data. Multi-locus phylogenetic analyses (ITS rDNA, nrLSU rDNA, RPB2, and TEF1-α) show that specimens recently collected in Yunnan Province, China are closely related to Gnomoniaceae. Both new species and known species were discovered repeatedly in their asexual developmental form exclusively on twigs of Rhododendron spp. (Ericaceae). This indicates a host specificity of Nagrajomyces spp. for species of Rhododendron.

Three new species of Candolleomyces (Agaricomycetes, Agaricales, Psathyrellaceae) from the Yanshan Mountains in China.

Three new species, Candolleomycesincanus, C.subcandolleanus and C.yanshanensis, were found and described from Yanshan Mountains in China. The identification is based on morphological observation combined with phylogenetic analysis of ITS-LSU-Tef1α-TUB2. This study enriched the species diversity of Candolleomyces in Yanshan Mountains and provided important data support for the systematic study of Candolleomyces in the future.

Morphological Characteristics and Phylogeny Reveal Six New Species in Russula Subgenus Russula (Russulaceae, Russulales) from Yanshan Mountains, North China.

Species of the genus Russula are key components of ectomycorrhizal ecosystems worldwide, some of which are famous edible fungi. Although many new species have been described in China, their diversity in North China is still poorly known. Based on the morphology observation of specimens and molecular phylogenetic analyses, combined with the current classification frame of Russula, six new species of Russula subgenus Russula are proposed from the Yanshan Mountains in northern Beijing and northern Hebei Province of China in this study: viz. Russula miyunensis (subsection Chamaeleontinae), R. plana (subsection Chamaeleontinae), R. sinoparva (subsection Puellarinae), R. sinorobusta (subsection Puellarinae), R. subversatilis (subsection Roseinae), and R. yanshanensis (subsection Puellarinae). This is the first report of the species of Russula subgenus Russula from the Yanshan Mountains. This study enriches the species diversity of Russula in North China and provides new data support for the systematic study of Russula in subsequent research, including research and development on edibility.