OBJECTIVETo obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus.

METHODSTotal RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smart trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'-RACE products.

RESULTSThis yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced.

CONCLUSIONThe transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

Amino Acid Sequence ; Base Sequence ; Blotting, Northern ; Citrinin ; biosynthesis ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Fungal Proteins ; chemistry ; genetics ; Gene Library ; Molecular Sequence Data ; Monascus ; genetics ; metabolism ; Mycelium ; genetics ; metabolism ; Pigments, Biological ; biosynthesis ; RNA, Messenger ; metabolism ; Sequence Alignment ; Sequence Analysis, DNA

OBJECTIVETo screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata.

METHODEndophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics.

RESULTFungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium.

CONCLUSIONEndophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

Acetylcholinesterase ; metabolism ; Acremonium ; genetics ; metabolism ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Chromatography, Thin Layer ; DNA, Fungal ; chemistry ; genetics ; DNA, Ribosomal ; chemistry ; genetics ; Diazonium Compounds ; metabolism ; Fungi ; classification ; genetics ; metabolism ; Huperzia ; microbiology ; Hydrolysis ; Naphthaleneacetic Acids ; metabolism ; Phylogeny ; RNA, Ribosomal, 18S ; classification ; genetics ; RNA, Ribosomal, 5.8S ; classification ; genetics ; Sequence Analysis, DNA

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

BACKGROUND: Next-generation sequencing (NGS) can detect many more microorganisms of a microbiome than traditional methods. This study aimed to analyze the vaginal microbiomes of Korean women by using NGS that included bacteria and other microorganisms. The NGS results were compared with the results of other assays, and NGS was evaluated for its feasibility for predicting vaginitis. METHODS: In total, 89 vaginal swab specimens were collected. Microscopic examinations of Gram staining and microbiological cultures were conducted on 67 specimens. NGS was performed with GS junior system on all of the vaginal specimens for the 16S rRNA, internal transcribed spacer (ITS), and Tvk genes to detect bacteria, fungi, and Trichomonas vaginalis. In addition, DNA probe assays of the Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis were performed. Various predictors of diversity that were obtained from the NGS data were analyzed to predict vaginitis. RESULTS: ITS sequences were obtained in most of the specimens (56.2%). The compositions of the intermediate and vaginitis Nugent score groups were similar to each other but differed from the composition of the normal score group. The fraction of the Lactobacillus spp. showed the highest area under the curve value (0.8559) in ROC curve analysis. The NGS and DNA probe assay results showed good agreement (range, 86.2-89.7%). CONCLUSIONS: Fungi as well as bacteria should be considered for the investigation of vaginal microbiome. The intermediate and vaginitis Nugent score groups were indistinguishable in NGS. NGS is a promising diagnostic tool of the vaginal microbiome and vaginitis, although some problems need to be resolved.
Area Under Curve ; Bacteria/*genetics/isolation & purification ; Bacterial Proteins/genetics ; Candida/*genetics/isolation & purification ; Female ; Fungal Proteins/genetics ; Gardnerella vaginalis/genetics/isolation & purification ; High-Throughput Nucleotide Sequencing ; Humans ; *Microbiota ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; ROC Curve ; Sequence Analysis, DNA ; Trichomonas vaginalis/genetics/isolation & purification ; Vagina/*microbiology ; Vaginitis/*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