Diversity of Trichoderma species associated with green mold contaminating substrates of Lentinula edodes and their interaction.

Introduction: The contamination of Trichoderma species causing green mold in substrates poses a significant obstacle to the global production of Lentinula edodes, adversely impacting both yield and quality of fruiting bodies. However, the diversity of Trichoderma species in the contaminated substrates of L. edodes (CSL) in China is not clear. The purpose of this study was to assess the biodiversity of Trichoderma species in CSL, and their interactions with L. edodes. Methods: A comprehensive two-year investigation of the biodiversity of Trichoderma species in CSL was conducted with 150 samples collected from four provinces of China. Trichoderma strains were isolated and identified based on integrated studies of phenotypic and molecular data. Resistance of L. edodes to the dominant Trichoderma species was evaluated in dual culture in vitro. Results: A total of 90 isolates were obtained and identified as 14 different Trichoderma species, including six new species named as Trichoderma caespitosus, T. macrochlamydospora, T. notatum, T. pingquanense, T. subvermifimicola, and T. tongzhouense, among which, T. atroviride, T. macrochlamydospora and T. subvermifimicola were identified as dominant species in the CSL. Meanwhile, three known species, namely, T. auriculariae, T. paraviridescens and T. subviride were isolated from CSL for the first time in the world, and T. paratroviride was firstly reported to be associated with L. edodes in China. Notebly, the in vitro evaluation of L. edodes resistance to dominant Trichoderma species showed strains of L. edodes generally possess poor resistance to Trichoderma contamination with L. edodes strain SX8 relatively higher resistant. Discussion: This study systematically investigated the diversity of Trichoderma species in the contaminated substrate of L. edodes, and a total of 31 species so far have been reported, indicating that green mold contaminated substrates of edible fungi were undoubtedly a biodiversity hotspot of Trichoderma species. Results in this study will provide deeper insight into the genus Trichoderma and lay a strong foundation for scientific management of the Trichoderma contamination in L. edodes cultivation.

Three New Trichoderma Species in Harzianum Clade Associated with the Contaminated Substrates of Edible Fungi.

Trichoderma is known worldwide as biocontrol agents of plant diseases, producers of enzymes and antibiotics, and competitive contaminants of edible fungi. In this investigation of contaminated substrates of edible fungi from North China, 39 strains belonging to 10 Trichoderma species isolated from four kinds of edible fungi were obtained, and three novel species belonging to the Harzianum clade were isolated from the contaminated substrates of Auricularia heimuer and Pholiota adipose. They were recognized based on integrated studies of phenotypic features, culture characteristics, and molecular analyses of RNA polymerase II subunit B and translation elongation factor 1-α genes. Trichoderma auriculariae was strongly supported as a separate lineage and differed from T. vermifimicola due to its larger conidia. Trichoderma miyunense was closely related to T. ganodermatigerum but differed due to its smaller conidia and higher optimum mycelial growth temperature. As a separate lineage, T. pholiotae was distinct from T. guizhouense and T. pseudoasiaticum due to its higher optimum mycelial growth temperature and larger conidia. This study extends the understanding of Trichoderma spp. contaminating substrates of edible fungi and updates knowledge of species diversity in the group.

Patterns of PCR Amplification Artifacts of the Fungal Barcode Marker in a Hybrid Mushroom.

The polymerase chain reaction (PCR) is widely used in modern biology and medicine. However, PCR artifacts can complicate the interpretation of PCR-based results. The internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster is the consensus fungal barcode marker and suspected PCR artifacts have been reported in many studies, especially for the analyses of environmental fungal samples. At present, the patterns of PCR artifacts in the whole fungal ITS region (ITS1+5.8S+ITS2) are not known. In this study, we analyzed the error rates of PCR at three template complexity levels using the divergent copies of ITS from the mushroom Agaricus subrufescens. Our results showed that PCR using the Phusion® High-Fidelity DNA Polymerase has a per nucleotide error rate of about 4 × 10-6 per replication. Among the detected mutations, transitions were much more frequent than transversions, insertions, and deletions. When divergent alleles were mixed as templates in the same reaction, a significant proportion (∼30%) of recombinant molecules were detected. The in vitro mixed-template results were comparable to those obtained from using the genomic DNA of the original mushroom specimen as template. Our results indicate that caution should be in place when interpreting ITS sequences from individual fungal specimens, especially those containing divergent ITS copies. Similar results could also happen to PCR-based analyses of other multicopy DNA fragments as well as single-copy DNA sequences with divergent alleles in diploid organisms.

Physiological Adjustments and Circulating MicroRNA Reprogramming Are Involved in Early Acclimatization to High Altitude in Chinese Han Males.

Background: Altitude acclimatization is a physiological process that restores oxygen delivery to the tissues and promotes oxygen use under high altitude hypoxia. High altitude sickness occurs in individuals without acclimatization. Unraveling the molecular underpinnings of altitude acclimatization could help understand the beneficial body responses to high altitude hypoxia as well as the altered biological events in un-acclimatized individuals. This study assessed physiological adjustments and circulating microRNA (cmiRNA) profiles in individuals exposed to high altitude, aiming to explore altitude acclimatization in humans. Methods: Ninety volunteers were enrolled in this study. Among them, 22 individuals provided samples for microRNA arrays; 68 additional individuals constituted the validation set. Un-acclimatized individuals were identified by the Lake Louise Scoring System. Thirty-three phenotypes were recorded pre- and post-exposure to high altitude, including stress hormones, lipid profiles, hematological indices, myocardial enzyme spectrum, and liver and kidney function related enzymes. CmiRNA expression profiles were assessed using miRCURYTM LNA Array (v.18.0) screening, with data validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then, associations of plasma microRNA expression with physiological adjustments were evaluated. The biological relevance of the main differentially expressed cmiRNAs was explored by bioinformatics prediction. Results: Nineteen of the 33 phenotypes were significantly altered during early altitude acclimatization, including hematological indices, lipid profiles, and stress hormones; meanwhile, 86 cmiRNAs (79 up-regulated and 7 down-regulated) showed differential expression with statistical significance. Among them, 32 and 25 microRNAs were strongly correlated with low-density lipoprotein-cholesterol and total cholesterol elevations, respectively. In addition, 22 microRNAs were closely correlated with cortisol increase. In un-acclimatized individuals, 55 cmiRNAs were up-regulated and 36 down-regulated, compared with acclimatized individuals. The HIF signaling pathway was suppressed in un-acclimatized individuals. Conclusion: Physiological adjustments, including the hematological system, stress hormones, and lipid molecules contributed to early altitude acclimatization, and showed strong correlations with cmiRNA reprogramming. Moreover, acclimatized and un-acclimatized individuals showed different cmiRNA profile. Suppression of the HIF-1 signaling pathway by microRNA regulation may play a key role in the pathogenesis of un-acclimatization with high altitude hypoxia.

Protection of Facial Nerves During Acoustic Neuroma Surgery.

The objective of this study was to summarize the experience about the protection of the facial nerve in surgery for acoustic neuroma surgery with the aim to improve the retention of facial nerve function and the quality of life. Forty-two patients with acoustic neuroma were recruited from the year 2010 to 2013. Using microsurgical techniques, the tumors were resected through the suboccipital approach over the posterior edge of the sigmoid sinus, and intraoperative electrophysiological monitoring of the facial nerve function was performed. The House-Brackmann (H-B) grading was used to evaluate the facial nerve function evaluation postoperatively. Total tumor resection was achieved in 32 cases, and partial resection in 10 cases, without any intraoperative deaths. Also facial nerves were retained in 35 of 42 cases (83.33 %). One week after surgery, the facial nerve H-B grading was grade I in 8 cases, grade II in 15 cases, grade III in 12 cases, grade IV in 6 cases, and grade V in 1 case. The key to improved protection of the facial nerve during acoustic neuroma surgery includes a complete understanding of the anatomy of the cerebellopontine angle, proper use of microsurgical techniques, and intraoperative electrophysiological monitoring of the status of facial nerve functions to avoid damage to the nerves.

A novel laccase with potent antiproliferative and HIV-1 reverse transcriptase inhibitory activities from mycelia of mushroom Coprinus comatus.

A novel laccase was isolated and purified from fermentation mycelia of mushroom Coprinus comatus with an isolation procedure including three ion-exchange chromatography steps on DEAE-cellulose, CM-cellulose, and Q-Sepharose and one gel-filtration step by fast protein liquid chromatography on Superdex 75. The purified enzyme was a monomeric protein with a molecular weight of 64 kDa. It possessed a unique N-terminal amino acid sequence of AIGPVADLKV, which has considerably high sequence similarity with that of other fungal laccases, but is different from that of C. comatus laccases reported. The enzyme manifested an optimal pH value of 2.0 and an optimal temperature of 60°C using 2,2'-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) as the substrate. The laccase displayed, at pH 2.0 and 37°C, K(m) values of 1.59 mM towards ABTS. It potently suppressed proliferation of tumor cell lines HepG2 and MCF7, and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) with an IC50 value of 3.46 µM, 4.95 µM, and 5.85 µM, respectively, signifying that it is an antipathogenic protein.

Coexistence of aneurysmal subarachnoid hemorrhage and surgically identified pituitary apoplexy: a case report and review of the literature.

INTRODUCTION: A ruptured aneurysm associated with a pituitary apoplexy is rare. We present the first case report of the coexistence of a ruptured posterior communicating aneurysm with a surgically discovered pituitary apoplexy where the pituitary apoplexy had not been diagnosed by a pre-operative computerized tomography scan. CASE PRESENTATION: A 31-year-old right-handed Chinese woman began to experience severe headache, vomiting and blurred vision which continued for two days. On admission to the hospital, a brain computerized tomography scan demonstrated a small amount of increased signal in the basal cisterns; no evidence of intrasellar and suprasellar lesions was seen. The appearance of her brain suggested aneurysmal subarachnoid hemorrhage. She had nuchal rigidity and reduced vision. There was no extra-ocular palsy and no other neurological deficit. Our patient had no stigmata of Cushing's syndrome or acromegaly. During an interview for further history, she reported normal menses and denied reduced vision.Cerebral digital subtraction angiography was subsequently performed, which revealed a 6mm left posterior communicating aneurysm. Urgent left pterional craniotomy was performed. The left ruptured posterior communicating artery aneurysm was completely dissected prior to clipping. At surgery, a suprasellar mass was discovered, the tumor bulging the diaphragma sella and projecting anteriorly under the chiasm raising suspicion of a pituitary tumor. The anterior part of the tumor capsule was opened and a necrotic tumor mixed with dark old blood was removed. The appearance suggested pituitary apoplexy.Histopathology revealed pituitary adenoma with evidence of hemorrhagic necrosis. Our patient made a good recovery. CONCLUSION: Our case report proves that pituitary apoplexy can be coexistent with the rupture of a posterior communicating aneurysm. This association should be considered when evaluating any case of aneurysm. A normal computerized tomography scan does not exclude pituitary apoplexy. Pre-operative magnetic resonance imaging interpretation is required if a pituitary apoplexy is suspected. Craniotomy allows a coexisting aneurysm and pituitary apoplexy to be simultaneously treated.

Purification and characterization of a novel lectin with antiphytovirus activities from the wild mushroom Paxillus involutus.

A novel lectin was isolated from the dried fruiting bodies of the wild mushroom Paxillus involutus. Isolation was conducted by anion exchange chromatography on DEAE-Cellulose, Q-Sepharose and gel filtration on Superdex 75 using a fast protein liquid chromatography (FPLC) system. This lectin had a molecular mass of 28 kDa and was composed of four identical subunits, each with a molecular mass of 7 kDa. N-terminal amino acid sequence of the P. involutus lectin was determined to be CTCAVFLNNTTVKS, which showed a low level of similarity to mushroom lectin sequences reported previously. The biochemical properties of this lectin were determined, and the hemagglutinating activity was inhibited by inulin and O-Nitrophenyl-ß-D-galacto-pyranoside. Additionally, Ca2+, Zn2+, Cd2+, Fe2+, and Al3+ inhibited its hemagglutinating activity, while Cu2+ promoted this activity. This lectin exhibited poor thermostability and was sensitive to HCl, but it had a high tolerance to NaOH exposure. In terms of biological properties, this lectin manifested antiphytovirus activity towards tobacco mosaic virus (TMV) with a 70.61% inhibition at a concentration of 200 µg/mL. This lectin was devoid of inhibitory activities toward pathogenic fungi and HIV-1 reverse transcriptase, and antiproliferative activities were observed in tumor cell lines including lung cancer A-549 and human colon cancer HCT-8 cells.

Evaluation of anti-quorum-sensing activity of fermentation metabolites from different strains of a medicinal mushroom, Phellinus igniarius.

AIMS: This study aimed to evaluate the different abilities of various Phellinus igniarius strains inhibiting quorum sensing (QS), to search for novel QS inhibitors from them and to analyze their inhibitory activity, with a view to their possible use in controlling infections. METHODS: The bioactive metabolites produced by P. igniarius cultures were tested for their abilities to inhibit QS-regulated behavior. All P. igniarius strains were cultured in potato-dextrose medium by large-scale submerged fermentation. The culture supernatant was condensed into 0.2-fold volumes by freeze drying. The condensed supernatant was sterilized by filtration through a 0.22-µm membrane filter and added to Chromobacterium violaceum CV026 cultures, which were used to monitor QS inhibition. Inhibitory activity was measured by quantifying violacein production using a microplate reader. RESULTS: The bioactive metabolites produced by 10 P. igniarius strains could inhibit violacein production, a QS-regulated behavior in C. violaceum. Furthermore, these strains could be roughly categorized into three groups on the basis of their inhibitory activities. CONCLUSIONS: P. igniarius strains can produce QS-inhibitory compounds and have different abilities to inhibit QS.

Cordysobin, a novel alkaline serine protease with HIV-1 reverse transcriptase inhibitory activity from the medicinal mushroom Cordyceps sobolifera.

A novel serine protease, designated as cordysobin, was purified from dried fruiting bodies of the mushroom Cordyceps sobolifera. The isolation procedure utilized ion exchange chromatography on DEAE-cellulose and SP-Sepharose followed by gel filtration on Superdex 75. The protease did not adsorb on DEAE-cellulose but bound to SP-Sepharose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protease resolved as a single band with an apparent molecular mass of 31 kDa. Its optimal pH was 10.0, and the optimal temperature was 65°C. The protease displayed a K(m) value of 0.41 µM and 13.44 µM·min⁻¹ using Suc-Leu-Leu-Val-Tyr-MCA as substrate at pH 10.0 and 37°C. Protease activity was enhanced by the Fe²âº ion at low concentration range of 1.25-10 mM and was strongly inhibited by Hg²âº up to 1.25 mM. The protease was strongly inhibited by chymostatin and phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease. It manifested significant inhibitory activity toward HIV-1 reverse transcriptase (RT) with an IC50 value of 8.2×10⁻³ µM, which is the highest anti-HIV-1 RT activity of reported mushroom proteins.

Inhibiting effect of bioactive metabolites produced by mushroom cultivation on bacterial quorum sensing-regulated behaviors.

AIMS: This study aimed to search for novel quorum sensing (QS) inhibitors from mushroom and to analyze their inhibitory activity, with a view to their possible use in controlling detrimental infections. METHODS: The bioactive metabolites produced by mushroom cultivation were tested for their abilities to inhibit QS-regulated behavior. All mushroom strains were cultivated in potato-dextrose medium by large-scale submerged fermentation. The culture supernatant was condensed into 0.2 vol by freeze-drying. The condensed supernatant was sterilized by filtration through a 0.22-µm membrane filter and added to Chromobacterium violaceum CV026 cultures, which were used to monitor QS inhibition. Inhibitory activity was measured by quantifying violacein production using a microplate reader. RESULTS: The results have revealed that, of 102 mushroom strains, the bioactive metabolites produced by 14 basidiomycetes were found to inhibit violacein production, a QS-regulated behavior in C. violaceum. CONCLUSIONS: Higher fungi can produce QS-inhibitory compounds.