Sexual Crossing, Chromosome-Level Genome Sequences, and Comparative Genomic Analyses for the Medicinal Mushroom Taiwanofungus Camphoratus (Syn. Antrodia Cinnamomea, Antrodia Camphorata).

Taiwanofungus camphoratus mushrooms are a complementary and alternative medicine for hangovers, cancer, hypertension, obesity, diabetes, and inflammation. Though Taiwanofungus camphoratus has attracted considerable biotechnological and pharmacological attention, neither classical genetic nor genomic approaches have been properly established for it. We isolated four sexually competent monokaryons from two T. camphoratus dikaryons used for the commercial cultivation of orange-red (HC1) and milky-white (SN1) mushrooms, respectively. We also sequenced, annotated, and comparatively analyzed high-quality and chromosome-level genome sequences of these four monokaryons. These genomic resources represent a valuable basis for understanding the biology, evolution, and secondary metabolite biosynthesis of this economically important mushrooms. We demonstrate that T. camphoratus has a tetrapolar mating system and that HC1 and SN1 represent two intraspecies isolates displaying karyotypic variation. Compared with several edible mushroom model organisms, T. camphoratus underwent a significant contraction in the gene family and individual gene numbers, most notably for plant, fungal, and bacterial cell-wall-degrading enzymes, explaining why T. camphoratus mushrooms are rare in natural environments, are difficult and time-consuming to artificially cultivate, and are susceptible to fungal and bacterial infections. Our results lay the foundation for an in-depth T. camphoratus study, including precise genetic manipulation, improvements to mushroom fruiting, and synthetic biology applications for producing natural medicinal products. IMPORTANCETaiwanofungus camphoratus (Tc) is a basidiomycete fungus that causes brown heart rot of the aromatic tree Cinnamomum kanehirae. The Tc fruiting bodies have been used to treat hangovers, abdominal pain, diarrhea, hypertension, and other diseases first by aboriginal Taiwanese and later by people in many countries. To establish classical genetic and genomic approaches for this economically important medicinal mushroom, we first isolated and characterized four sexually competent monokaryons from two dikaryons wildly used for commercial production of Tc mushrooms. We applied PacBio single molecule, real-time sequencing technology to determine the near-completed genome sequences of four monokaryons. These telomere-to-telomere and gapless haploid genome sequences reveal all genomic variants needed to be studied and discovered, including centromeres, telomeres, retrotransposons, mating type loci, biosynthetic, and metabolic gene clusters. Substantial interspecies diversities are also discovered between Tc and several other mushroom model organisms, including Agrocybe aegerita, Coprinopsis cinerea, and Schizophyllum commune, and Ganoderma lucidum.

Fluconazole induces rapid high-frequency MTL homozygosis with microbiological polymorphism in Candida albicans.

BACKGROUND: Candida albicans, a common fungal pathogen that can cause opportunistic infections, is regarded as an apparently asexual, diploid fungus. A parasexual cycle was previously found between homozygotes with opposite mating type-like loci (MTLa/α). Fluconazole-resistant strains had a higher proportion of MTL homozygotes, whereas MTL homozygous C. albicans was found in only about 3.2% of clinical strains. MTL heterozygotes had a low frequency (1.4 × 10-4) of white-opaque switching to MTL homozygotes in nature. METHODS: Here, a reference C. albicans strain (SC5314) was used in a fluconazole-induced assay to obtain standard opaque MTL homozygous strains and first-generation daughter strains from the fluconazole inhibition zone. Further separation methods were employed to produce second- and third-generation daughter strains. Polymerase chain reaction analysis based on MTL genes was used to define MTL genotypes, and microscopic observations, a flow-cytometric assay, and an antifungal E-test were used to compare microbiological characteristics. RESULTS: MTL homozygotes were found at a high frequency (17 of 35; 48.6%) in fluconazole-induced first-generation daughter strains, as were morphological polymorphisms, decreased DNA content, and modified antifungal drug susceptibility. High-frequency MTL homozygosity was identified inside the fluconazole inhibition zone within 24 hours. The DNA content of fluconazole-induced daughter strains was reduced compared with their progenitor SC5314 and standard MTL homozygous strains. CONCLUSION: Treatment with fluconazole, commonly used to treat invasive candidiasis, inhibited the growth of C. albicans and altered its microbiological characteristics. Our results suggest that fluconazole treatment induces the high frequency of loss of heterozygosity and microbiological polymorphism in C. albicans.

Oxygen-implanted induced formation of oxide layer enhances blood compatibility on titanium for biomedical applications.

Titanium dioxide (TiO2) layers were prepared on a Ti substrate by using oxygen plasma immersion ion implantation (oxygen PIII). The surface chemical states, structure, and morphology of the layers were studied using X-ray photoelectron spectroscopy, X-ray diffraction, Raman microscopy, atomic force microscopy and scanning electron microscope. The mechanical properties, such as the Young's modulus and hardness, of the layers were investigated using nanoindentation testing. The Ti(4+) chemical state was determined to be present on oxygen-PIII-treated surfaces, which consisted of nanocrystalline TiO2 with a rutile structure. Compared with Ti substrates, the oxygen-PIII-treated surfaces exhibited decreased Young's moduli and hardness. Parameters indicating the blood compatibility of the oxygen-PIII-treated surfaces, including the clotting time and platelet adhesion and activation, were studied in vitro. Clotting time assays indicated that the clotting time of oxygen-PIII-treated surfaces was longer than that of the Ti substrate, which was associated with decreased fibrinogen adsorption. In conclusion, the surface characteristics and the blood compatibility of Ti implants can be modified and improved using oxygen PIII.

The Ethanolic Extract of Taiwanofungus camphoratus (Antrodia camphorata) Induces Cell Cycle Arrest and Enhances Cytotoxicity of Cisplatin and Doxorubicin on Human Hepatocellular Carcinoma Cells.

Taiwanofungus camphoratus (synonym Antrodia camphorata) is a widely used medicinal fungus in the folk medicine of Taiwan with several pharmacological features such as anti-inflammatory, liver protection, antihypertensive, and antioxidative activities. The ethanolic extract of T. camphoratus (TCEE) which contains abundant bioactive compounds including triterpenoids and polysaccharides also has antitumor effects in various human cancer cell lines. The aims of this study are to clarify the antitumor effects of TCEE on human hepatocellular carcinoma cells and also evaluate the combination drug effects with conventional chemotherapy agents, cisplatin and doxorubicin. In the present study, the TCEE treatment induced cell cycle arrest and suppressed cell growth on both Hep3B and HepJ5 cells. Expression of cell cycle inhibitors, P21 and P27, and activation of apoptosis executer enzyme, caspase-3, were also induced by TCEE. In combination with the chemotherapy agents, TCEE treatment further enhanced the tumor suppression efficiency of cisplatin and doxorubicin. These results together suggested that TCEE is a potential ingredient for developing an integrated chemotherapy for human liver cancer.

Short-term exposure to fluconazole induces chromosome loss in Candida albicans: an approach to produce haploid cells.

Candida albicans is considered to be an obligate diploid fungus. Here, we describe an approach to isolate aneuploids or haploids induced by the short-term (12-16 h) exposure of diploid reference strains SC5314 and CAI4 to the most commonly used antifungal drug, fluconazole, followed by repeated single-cell separation among small morphologically distinct colonies in the inhibition zone. The isolated strains had altered cell morphology and LOH events in the MTL and other marker alleles of the analyzed loci at 8 chromosomes of C. albicans with decreased DNA content. The present study employed next-generation sequencing (NGS) combined flow cytometry analysis of the DNA content to analyze the haploid, autodiploid, and aneuploid strains that arose from the fluconazole treatment instead of using the conventional single nucleotide polymorphism/comparative genome hybridization (SNP/CGH) method. A multiple-alignment tool was also developed based on sequenced data from NGS to establish haplotype mapping for each chromosome of the selected strains. These findings revealed that C. albicans experiences 'concerted chromosome loss' to form strains with homozygous alleles and that it even has a haploid status after short-term exposure to fluconazole. Additionally, we developed a new platform to analyze chromosome copy number using NGS.