In this study, we employed a combination of genome mining and heteronuclear single quantum coherence (HSQC)-based small molecule accurate recognition technology (SMART) technology to search for fernane-type triterpenoids. Initially, potential endophytic fungi were identified through genome mining. Subsequently, fine fractions containing various fernane-type triterpenoids were selected using HSQC data collection and SMART prediction. These triterpenoids were then obtained through targeted isolation and identification. Finally, their antifungal activity was evaluated. As a result, three fernane-type triterpenoids, including two novel compounds, along with two new sesquiterpenes and four known compounds were isolated from one potential strain, Diaporthe discoidispora. Their structures were elucidated through analysis of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopic data. The absolute configurations were determined using single-crystal X-ray diffraction analysis and electron capture detector (ECD) analysis. Compound 3 exhibited moderate antifungal activity against Candida albicans CMCC 98001 and Aspergillus niger.
Triterpenes/isolation & purification* ; Antifungal Agents/isolation & purification* ; Molecular Structure ; Candida albicans/drug effects* ; Ascomycota/genetics* ; Magnetic Resonance Spectroscopy ; Aspergillus niger/drug effects* ; Genome, Fungal ; Microbial Sensitivity Tests

Two novel diketopiperazines (1 and 5), along with ten known compounds (2-4, 6-12) demonstrating significant skin inflammation inhibition, were isolated from a marine-derived fungus identified as Aspergillus sp. FAZW0001. The structural elucidation and configurational reassessments of compounds 1-5 were established through comprehensive spectral analyses, with their absolute configurations determined via single crystal X-ray diffraction using Cu Kα radiation, Marfey's method, and comparison between experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 1, 2, and 8 exhibited significant anti-inflammatory activities in Propionibacterium acnes (P. acnes)-induced human monocyte cell lines. Compound 8 demonstrated the ability to down-regulate interleukin-1β (IL-1β) expression by inhibiting Toll-like receptor 2 (TLR2) expression and modulating the activation of myeloid differentiation factor 88 (MyD88), mitogen-activated protein kinase (MAPK), and nuclear factor κB (NF-κB) signaling pathways, thus reducing the cellular inflammatory response induced by P. acnes. Additionally, compound 8 showed the capacity to suppress mitochondrial reactive oxygen species (ROS) production and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation, thereby reducing IL-1β maturation and secretion. A three-dimensional quantitative structure-activity relationships (3D-QSAR) model was applied to compounds 5-12 to analyze their anti-inflammatory structure-activity relationships.
Humans ; Aspergillus/chemistry* ; Diketopiperazines/isolation & purification* ; Anti-Inflammatory Agents/isolation & purification* ; Interleukin-1beta/genetics* ; Toll-Like Receptor 2/immunology* ; Propionibacterium acnes/drug effects* ; NF-kappa B/genetics* ; Molecular Structure ; Myeloid Differentiation Factor 88/immunology* ; Monocytes/immunology* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Cell Line

OBJECTIVE: A strain of Aspergillus niger (A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain (FA-WB) were evaluated. METHODS: Molecular identification techniques based on PCR analysis of specific genomic sequences were conducted; antioxidant ability was examined using oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA) assays, and erythrocyte hemolysis assays. RAW264.7 cells were used as a model to detect anti-inflammatory activity. RESULTS: The filamentous fungal isolate was identified to be A. niger. ORAC and CAA assay showed that FA-WB had better antioxidant activity than that of the ferulic acid standard. The erythrocyte hemolysis assay results suggested that FA-WB could attenuate AAPH-induced oxidative stress through inhibition of reactive oxy gen species (ROS) generation. FA-WB could significantly restore the AAPH-induced increase in intracellular antioxidant enzyme activities to normal levels as well as inhibit the intracellular malondialdehyde formation. TNF-a, IL-6, and NO levels indicated that FA-WB can inhibit the inflammation induced by lipopolysaccharide (LPS). CONCLUSION Ferulic acid released from wheat bran by a new strain of A. niger had good anti-inflammatory activity and better antioxidant ability than standard ferulic acid.
Animals ; Anti-Inflammatory Agents ; metabolism ; pharmacology ; Antioxidants ; metabolism ; pharmacology ; Aspergillus niger ; genetics ; isolation & purification ; metabolism ; Coumaric Acids ; metabolism ; pharmacology ; DNA, Fungal ; analysis ; Dietary Fiber ; microbiology ; Erythrocytes ; drug effects ; metabolism ; Fermentation ; Hep G2 Cells ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Mice ; RAW 264.7 Cells ; Sheep ; Tumor Necrosis Factor-alpha ; metabolism

Chemical examination of an EtOAc extract of cultured Aspergillus versicolor fungus from deep-sea sediments resulted in the isolation of four xanthones, eight anthraquinones and five alkaloids, including a new xanthone, oxisterigmatocystin D (1) and a new alkaloid, aspergillusine A (13). High resolution electron impact mass spectrometry (HR-EI-MS), FT-IR spectroscopy, and NMR techniques were used to elucidate the structures of these compounds, and the absolute configuration of compound 1 was established by its NMR features and coupling constant. Furthermore, the biosynthesis pathway of these xanthones and anthraquinones were deduced, and their antioxidant activity and cytotoxicity in human cancer cell lines (HTC-8, Bel-7420, BGC-823, A549, and A2780) were evaluated. The trolox equivalent antioxidant capacity (TEAC) assay indicated most of the xanthones and anthraquinones possessing moderate antioxidant activities. The Nrf2-dependent luciferase reporter gene assay revealed that compounds 6, 7, 9, and 12 potentially activated the expression of Nrf2-regulated gene. In addition, compounds 5 and 11 showed weak cytotoxicity on A with the IC values of 25.97 and 25.60 μmol·L, respectively.
Anthraquinones ; Antioxidants ; chemistry ; isolation & purification ; metabolism ; pharmacology ; Aspergillus ; chemistry ; genetics ; isolation & purification ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Gene Expression ; drug effects ; Humans ; Magnetic Resonance Spectroscopy ; Molecular Structure ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Seawater ; microbiology ; Spectroscopy, Fourier Transform Infrared ; Xanthones ; chemistry ; isolation & purification ; metabolism ; pharmacology

BACKGROUND: We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. METHODS: A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and beta-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. RESULTS: ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by beta-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of > or =2 microg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was < or =75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). CONCLUSIONS: Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.
Amphotericin B/*pharmacology ; Antifungal Agents/*pharmacology ; Aspergillus/*drug effects/isolation & purification ; DNA, Fungal/chemistry/genetics/metabolism ; Hospitals ; Humans ; Microbial Sensitivity Tests ; Mycoses/diagnosis/microbiology ; Republic of Korea ; Sequence Analysis, DNA ; Tubulin/genetics

Monitoring the response to therapy for invasive aspergillosis (IA) is essential for the management of patients with hematologic diseases. We evaluated the correlation between the outcome of real-time nucleic acid sequence-based amplification (RTi-NASBA) for Aspergillus 18S rRNA and the clinical outcome of IA. A total of 157 serum samples from 29 patients with IA were tested for RTi-NASBA. The treatment response and mortality were compared with the NASBA outcome (whether the NASBA value was converted to negative or not) at 12 weeks after the start of antifungal therapy. At 12 weeks, there was a moderate correlation between the treatment failure and persistently positive NASBA (kappa = 0.482; P = 0.019). Deaths attributable to IA were more prevalent in patients without negative conversion of NASBA than in those with negative conversion (50% vs 5%; P = 0.013). Significant factors of treatment failure at 12 weeks were the status of hematologic disease (nonremission; P = 0.041) and the NASBA outcome (failure of negative conversion; P = 0.024). Survival was significantly better in patients with negative conversion of NASBA than those with persistently positive values (P = 0.036). This study suggests that the serial monitoring of RTi-NASBA could be useful for prediction of the clinical outcome in hematologic patients with IA.
Adolescent ; Adult ; Aged ; Antifungal Agents/therapeutic use ; Aspergillosis/*diagnosis/drug therapy/microbiology/mortality ; Aspergillus/*genetics/isolation & purification ; Base Sequence ; Female ; Humans ; Lung/microbiology ; Male ; Middle Aged ; Predictive Value of Tests ; RNA, Ribosomal, 18S/analysis ; *Real-Time Polymerase Chain Reaction ; Retrospective Studies ; Sputum/microbiology ; Survival Rate

BACKGROUND: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. METHODS: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the beta-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. RESULTS: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and beta-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of beta-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. CONCLUSIONS: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.
Aspergillus/genetics/isolation & purification ; DNA, Fungal/analysis/isolation & purification ; Databases, Genetic ; Fungi/genetics/*isolation & purification ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 28S/*genetics ; Sequence Analysis, DNA ; Trichophyton/genetics/isolation & purification ; Tubulin/*genetics

OBJECTIVETo evaluate the feasibility of PCR/reverse line blot hybridization (RLB) assay in the detection and identification of clinical pathogens in fungal sinusitis (FS).

METHODSTwenty-six formalin-fixed and paraffin-embedded tissues and 8 fresh tissues of FS were collected from Beijing Tongren Hospital, Capital Medical University from May 2009 to February 2010. Pathological examination, fungal culture and ITS2 region sequencing were carried out. The DNA of all samples was extracted by standard procedure and fungal universal primers ITS3 and ITS4 were used for PCR amplification of all tissues. Then the amplified products were used for RLB with five fungal species-specific probes. The results of PCR/RLB were compared with ITS region sequencing, fungal culture and pathological examination.

RESULTSFor the biopsy tissues, fungal cultures were positive in 14 cases (41.2%); pathologic examination demonstrated fungal hyphae in all cases; ITS2 region sequencing was successful in 16 cases (47.1%); PCR/RLB showed A. flavus in 14 cases, A. fumigatus in 10 cases, A. niger in four cases, A. nidulans in one case, A. flavus and A. fumigatus in three cases, and A. fumigatus and A. niger in two cases.

CONCLUSIONSThe PCR/RLB assay is suitable for rapid and accurate detection and identification of the pathogenic fungus of FS. Compared with the conventional fungal culture and microscopy, pathologic examination and DNA sequencing, the PCR/RLB has the advantages of more economy, time saving, and higher sensitivity, specificity and throughput.

Aspergillus ; classification ; genetics ; isolation & purification ; Aspergillus flavus ; genetics ; isolation & purification ; Aspergillus fumigatus ; genetics ; isolation & purification ; Aspergillus niger ; genetics ; isolation & purification ; DNA Primers ; DNA, Fungal ; genetics ; Humans ; Mycoses ; diagnosis ; microbiology ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Sinusitis ; diagnosis ; microbiology

OBJECTIVETo isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities.

METHODFungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA.

RESULTNinety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum.

CONCLUSIONEndophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.

Anti-Infective Agents ; metabolism ; pharmacology ; Aspergillus fumigatus ; drug effects ; Bacillus subtilis ; drug effects ; Base Sequence ; Biodiversity ; Candida albicans ; drug effects ; China ; Cryptococcus neoformans ; drug effects ; DNA, Fungal ; chemistry ; isolation & purification ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Dendrobium ; microbiology ; physiology ; Endophytes ; classification ; genetics ; isolation & purification ; physiology ; Escherichia coli ; drug effects ; Fungi ; classification ; genetics ; isolation & purification ; physiology ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; microbiology ; physiology ; Plant Stems ; microbiology ; physiology ; Sequence Alignment ; Sequence Analysis, DNA ; Staphylococcus aureus ; drug effects

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.
Aspergillus flavus ; enzymology ; Cloning, Molecular ; Codon ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mutation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Urate Oxidase ; biosynthesis ; genetics ; isolation & purification ; Uric Acid ; metabolism