Isolation, Culture and Characterization of Hirsutella sinensis Mycelium from Caterpillar Fungus Fruiting Body

The caterpillar fungus Ophiocordyceps sinensis (previously called Cordyceps sinensis) has been used for centuries in Asia as a tonic to improve health and longevity. Recent studies show that O. sinensis produces a wide range of biological effects on cells, laboratory animals and humans, including anti-fatigue, anti-infection, anti-inflammatory, antioxidant, and anti-tumor activities. In view of the rarity of O. sinensis fruiting bodies in nature, cultivation of its anamorph mycelium represents a useful alternative for large-scale production. However, O. sinensis fruiting bodies harvested in nature harbor several fungal contaminants, a phenomenon that led to the isolation and characterization of a large number of incorrect mycelium strains. We report here the isolation of a mycelium from a fruiting body of O. sinensis and we identify the isolate as O. sinensis’ anamorph (also called Hirsutella sinensis) based on multi-locus sequence typing of several fungal genes (ITS, nrSSU, nrLSU, RPB1, RPB2, MCM7, β-tubulin, TEF-1α, and ATP6). The main characteristics of the isolated mycelium, including its optimal growth at low temperature (16°C) and its biochemical composition, are similar to that of O. sinensis fruiting bodies, indicating that the mycelium strain characterized here may be used as a substitute for the rare and expensive O. sinensis fruiting bodies found in nature

Ganoderma Lucidum Stimulates Autophagy-Dependent Longevity Pathways in Caenorhabditis Elegans and Human Cells

The medicinal fungus Ganoderma lucidum is used as a dietary supplement and health tonic, but whether it affects longevity remains unclear. We show here that a water extract of G. lucidum mycelium extends lifespan of the nematode Caenorhabditis elegans. The G. lucidum extract reduces the level of fibrillarin (FIB-1), a nucleolar protein that correlates inversely with longevity in various organisms. Furthermore, G. lucidum treatment increases expression of the autophagosomal protein marker LGG-1, and lifespan extension is abrogated in mutant C. elegans strains that lack atg-18, daf-16, or sir-2.1, indicating that autophagy and stress resistance pathways are required to extend lifespan. In cultured human cells, G. lucidum increases concentrations of the LGG-1 ortholog LC3 and reduces levels of phosphorylated mTOR, a known inhibitor of autophagy. Notably, low molecular weight compounds (\u3c10 kDa) isolated from the G. lucidum water extract prolong lifespan of C. elegans and the same compounds induce autophagy in human cells. These results suggest that G. lucidum can increase longevity by inducing autophagy and stress resistance

Myths and Realities Surrounding the Mysterious Caterpillar Fungus

The caterpillar fungus Ophiocordyceps sinensis is a medicinal mushroom increasingly used as a dietary supplement for various health conditions, including fatigue, chronic inflammation, and male impotence. Here, we propose strategies to address the existing challenges related to the study and commercial production of this mysterious fungus

Clinical Characteristics and In-Hospital Outcomes in Dialysis Patients with Septic Arthritis

Background and Objectives: Septic arthritis is a medical emergency associated with high morbidity and mortality. The incidence rate of septic arthritis among dialysis patients is higher than the general population, and dialysis patients with bacteremia frequently experience adverse outcomes. The aim of this study was to identify the clinical features and risk factors for longer hospital length of stay (LOS), positive blood culture, and in-hospital mortality in dialysis patients with septic arthritis. Materials and Methods: The medical records of 52 septic arthritis dialysis patients admitted to our hospital from 1 January 2009 to 31 December 2020 were analyzed. The primary outcomes were bacteremia and in-hospital mortality. Variables were compared, and risk factors were evaluated using linear and logistic regression models. Results: Twelve (23.1%) patients had positive blood cultures. A tunneled cuffed catheter for dialysis access was used in eight (15.4%) patients, and its usage rate was significantly higher in patients with positive blood culture than in those with negative blood culture (41.7 vs. 7.5%, p = 0.011). Fever was present in 15 (28.8%) patients, and was significantly more frequent in patients with positive blood culture (58.3 vs. 20%, p = 0.025). The most frequently involved site was the hip (n = 21, 40.4%). The most common causative pathogen was Gram-positive cocci, with MRSA (n = 7, 58.3%) being dominant. The mean LOS was 29.9 ± 25.1 days. The tunneled cuffed catheter was a significant predictor of longer LOS (Coef = 0.49; Cl 0.25–0.74; p p = 0.037) and tunneled cuffed catheter (OR = 7.60; Cl 1.31–44.02; p = 0.024). The predictor of mortality was tunneled cuffed catheter (OR = 14.33; Cl 1.12–183.18; p = 0.041). Conclusions: In the dialysis population, patients with tunneled cuffed catheter for dialysis access had a significantly longer hospital LOS. Tunneled cuffed catheter and fever were independent predictors of positive blood culture, and tunneled cuffed catheter was the predictor of in-hospital mortality. The recognition of the associated factors allows for risk stratification and determination of the optimal treatment plan in dialysis patients with septic arthritis

Pinicolol B from Antrodia cinnamomea induces apoptosis of nasopharyngeal carcinoma cells

Ethnopharmacological relevance: The medicinal mushroom Antrodia cinnamomea possesses anticancer properties but the active compounds responsible for these effects are mostly unknown. Aim of the study: We aimed to identify novel A. cinnamomea compounds that produce cytotoxic effects on cancer cells. Materials and methods: Using ethanol extraction and chromatography, we isolated the lanostanoid compound lanosta-7,9(11),24-trien-3β,15α,21-triol (1) from cultured A. cinnamomea mycelium. Cytotoxicity and pro-apoptotic effects of compound 1 were evaluated using the MTS assay and flow cytometry analysis, respectively. Results: Compound 1 produced cytotoxic effects on the nasopharyngeal carcinoma cell lines TW02 and TW04, with IC50 values of 63.3 and 115.0μM, respectively. On the other hand, no cytotoxic effects were observed on non-tumorigenic nasopharyngeal epithelial cells (NP69). In addition, compound 1 induced apoptosis in TW02 and TW04 cells as revealed by flow cytometry analysis. Conclusions: Our results demonstrate for the first time the presence of pinicolol B in A. cinnamomea mycelium and suggest that this compound may contribute to the anticancer effects of A. cinnamomea. Pinicolol B from Antrodia cinnamomea induces apoptosis of nasopharyngeal carcinoma cells

Isolation, Culture and Characterization of Hirsutella sinensis Mycelium from Caterpillar Fungus Fruiting Body

The caterpillar fungus Ophiocordyceps sinensis (previously called Cordyceps sinensis) has been used for centuries in Asia as a tonic to improve health and longevity. Recent studies show that O. sinensis produces a wide range of biological effects on cells, laboratory animals and humans, including anti-fatigue, anti-infection, anti-inflammatory, antioxidant, and anti-tumor activities. In view of the rarity of O. sinensis fruiting bodies in nature, cultivation of its anamorph mycelium represents a useful alternative for large-scale production. However, O. sinensis fruiting bodies harvested in nature harbor several fungal contaminants, a phenomenon that led to the isolation and characterization of a large number of incorrect mycelium strains. We report here the isolation of a mycelium from a fruiting body of O. sinensis and we identify the isolate as O. sinensis’ anamorph (also called Hirsutella sinensis) based on multi-locus sequence typing of several fungal genes (ITS, nrSSU, nrLSU, RPB1, RPB2, MCM7, β-tubulin, TEF-1α, and ATP6). The main characteristics of the isolated mycelium, including its optimal growth at low temperature (16°C) and its biochemical composition, are similar to that of O. sinensis fruiting bodies, indicating that the mycelium strain characterized here may be used as a substitute for the rare and expensive O. sinensis fruiting bodies found in nature

Isolation, Culture and Characterization of Hirsutella sinensis Mycelium from Caterpillar Fungus Fruiting Body.

The caterpillar fungus Ophiocordyceps sinensis (previously called Cordyceps sinensis) has been used for centuries in Asia as a tonic to improve health and longevity. Recent studies show that O. sinensis produces a wide range of biological effects on cells, laboratory animals and humans, including anti-fatigue, anti-infection, anti-inflammatory, antioxidant, and anti-tumor activities. In view of the rarity of O. sinensis fruiting bodies in nature, cultivation of its anamorph mycelium represents a useful alternative for large-scale production. However, O. sinensis fruiting bodies harvested in nature harbor several fungal contaminants, a phenomenon that led to the isolation and characterization of a large number of incorrect mycelium strains. We report here the isolation of a mycelium from a fruiting body of O. sinensis and we identify the isolate as O. sinensis' anamorph (also called Hirsutella sinensis) based on multi-locus sequence typing of several fungal genes (ITS, nrSSU, nrLSU, RPB1, RPB2, MCM7, β-tubulin, TEF-1α, and ATP6). The main characteristics of the isolated mycelium, including its optimal growth at low temperature (16°C) and its biochemical composition, are similar to that of O. sinensis fruiting bodies, indicating that the mycelium strain characterized here may be used as a substitute for the rare and expensive O. sinensis fruiting bodies found in nature

5.8S-ITS rDNA phylogenetic tree of <i>Ophiocordyceps</i> species.

<p>The evolutionary relationship of <i>Ophiocordyceps</i> 5.8S-ITS rDNA genes was determined using the neighbor-joining method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168734#pone.0168734.ref032" target="_blank">32</a>]. Evolutionary distances were assessed using the maximum composite likelihood (MCL) method. The bootstrap consensus tree, which represents the evolutionary relationship of the analyzed taxa, was inferred from 500 replicates as before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168734#pone.0168734.ref027" target="_blank">27</a>]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. Evolutionary distances (i.e., number of base substitutions per site) were computed using the maximum composite likelihood method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168734#pone.0168734.ref024" target="_blank">24</a>].</p

Culture of <i>H</i>. <i>sinensis</i> mycelium derived from <i>O</i>. <i>sinensis</i> fruiting body.

<p>(A) <i>O</i>. <i>sinensis</i> fruiting body or stroma (top) protruding from the shell of a caterpillar insect (bottom) was obtained in the Naqu prefecture in Tibet. HSM strain CGB 999335 was isolated from a similar fruiting body. (B) Colony of HSM strain CGB 999335 cultured for 28 days at 18°C on PDA agar. (C) CGB 999335 mycelium observed under optical microscopy. (D) Sterile FM1 liquid medium used to culture CGB 999335 mycelium in the present study (left tube; containing 1.2% of yeast extract as a source of nitrogen) and sterile liquid 1.2% (w/v) soybean broth commonly used in other laboratories (tube on the right). Notice the pellet of undissolved powder in the tube on the right. See the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168734#sec002" target="_blank">Methods</a> section for more details. (E) Dark-field optical microscopy image of CGB 999335 mycelium cultured in FM1 medium. (F) CGB 999335 mycelium cultured in soybean broth seen in D (tube on the right). Undissolved, brown material can be seen among mycelial cells.</p

Culture of <i>H</i>. <i>sinensis</i> mycelium at various pH.

<p>CGB 999335 mycelium was cultured in liquid FM1 medium at 16°C with mixing. Prior to culture, the pH of the culture medium was adjusted to the indicated value by adding 1 M HCl or NaOH. After five days of culture, mycelium cells were obtained by centrifugation, followed by drying and measurement of biomass weight.</p