Bioinformatics Analysis, Expression Profiling, and Functional Characterization of Heat Shock Proteins in Wolfi-poria cocos.

Heat shock proteins (HSPs) play critical roles in regulating different mechanisms under high-temperature conditions. HSPs have been identified and well-studied in different plants. However, there is a lack of information about their genomic organization and roles in medicinal plants and fungi, especially in Wolfi-poria cocos (W. cocos). We identified sixteen heat shock proteins (HSPs) in W. cocos and analyzed in terms of phylogenetic analysis, gene structure, motif distribution patterns, physiochemical properties, and expression comparison in different strains. Based on phylogenetic analysis, HSPs were divided into five subgroups (WcHSP100, WcHSP90, WcHSP70, WcHSP60, and WcsHSP). Subgroups WcHSP100s, WcHSP90s, WcHSP70s, WcHSP60, and WcsHSPs were further divided into 3, 2, 3, 1, and 6 subfamilies, respectively. Moreover, the expression profiling of all HSP genes in five strains of W. cocos under different temperature extremes revealed that expression of most HSPs were induced by high temperature. However, every subfamily showed different expression suggesting distinctive role in heat stress tolerance. WcHSP70-4, WcHSP90-1, and WcHSP100-1 showed the highest response to high temperature stress. Heterologous expression of WcHSP70-4, WcHSP90-1, and WcHSP100-1 genes in Escherichia coli enhanced survival rate of E. coli during heat stress. These findings suggest the role of W. cocos heat shock genes in the high temperature stress tolerance.

Cloning, characterization, and heterologous expression of a candidate Hirudin gene from the salivary gland transcriptome of Hirudo nipponia.

Hirudin is a pharmacologically active substance in leeches with potent blood anticoagulation properties. Although recombinant hirudin production isolated from Hirudo medicinalis Linnaeus and Hirudinaria manillensis Lesson is known, to our knowledge, this study is the first to report recombinant hirudin expression and production from Hirudo nipponia Whitman. Thus, the present study aimed to clone and characterize the full-length cDNA of a candidate hirudin gene (c16237_g1), which is localized on the salivary gland transcriptome of H. nipponia, and further evaluate its recombinant production using a eukaryotic expression system. The 489-bp cDNA possessed several properties of the hirudin "core" motifs associated with binding to the thrombin catalytic pocket. A fusion expression vector (pPIC9K-hirudin) was constructed and successfully transformed into Pichia pastoris strain GS115 via electroporation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blot analysis confirmed hirudin expression. The recombinant protein was expressed with a yield of 6.68 mg/L culture. Mass spectrometry analysis further confirmed target protein expression. The concentration and antithrombin activity of purified hirudin were 1.67 mg/mL and 14,000 ATU/mL, respectively. These findings provide a basis for further elucidating the molecular anticoagulation mechanism of hirudin, and address China's growing market demand for engineered H. nipponia-derived hirudin and hirudin-based drugs.

Detection of the pathogenic fungus Cordyceps farinosa in the Thitarodes armoricanus soil-rearing environment based on nucleic acid targets.

Cordyceps farinosa, an entomopathogenic fungus, infects and leads to high mortality of Thitarodes armoricanus larvae, which die soon after the infection of C. farinose, usually before the colonization of Ophiocordyceps sinensis owing to competitive inhibition and fruiting body formation. Therefore, monitoring C. farinosa in the O. sinensis cultivation environment is critical for minimizing the C. farinosa infection-induced losses. In this study, we initially designed a PCR primer pair (Tar-1F/Tar-1R) through open reading frame prediction and homology comparison of the C. farinosa genome sequence. This primer pair can detect both C. farinosa and Samsoniella hepiali. To further distinguish, primers (ITS5-172/ITS4-95) were then designed to selectively amplify the large ribosomal subunit sequences in the C. farinosa genome. All these primers were applied in combination for detection of C. farinosa in soil samples. The sensitivity reached a detection limit of 1 × 106 spores/g soil. In addition, these primers can detect the presence of C. farinosa in dead T. armoricanus larval samples. This newly established rapid detection method provides important information for C. farinosa control during O. sinensis cultivation.

Fractal characteristics of overburden fissures in shallow thick coal seam mining in loess gully areas.

The problems of water scarcity and ecological fragility are common in the loess gully area. To research the distribution and evolution of the overburden fissures and quantitatively analyze them have certain theoretical and engineering significance for realizing the evaluation of overburden damage degree and safe and green mining. This paper takes the 6102 working face of Chuancao Gedan Coal Mine as the engineering background. The development law and distribution characteristics of overburden fissures caused by the mining of shallow coal seams in the loess gully area were studied by the combination of physical similarity simulation, numerical similarity simulation and fractal theory. The results show that the fractal dimension change of the overburden fissures caused by the shallow mining of coal seam groups in the loess gully area can be divided into three stages during the mining process of the working face. Repeated mining causes the activation and development of overburden fissures, the fractal dimension increases significantly, and the regularity of changes weakens. The magnitude of the stress near the working face and the fluctuation times of the stress in the goaf have an influence on the change of the fractal dimension of the overburden fissures. According to the development angle and the fractal dimension of the overburden fissures, the overburden rock above the goaf is divided into the collapse fissure area, the compaction fissure area, and the vertical fissure area. Overburden fissures develop violently in the vertical fissure area, the overburden fissures in the compaction fissure area are mostly transverse fissures, and the overburden fissures in the caving fissure area are irregular.

Study on generation, migration and accumulation of CO in the mining goaf of shallow-buried close distance coal seam group.

There is complex air leakage in the mining of shallow buried close distance coal seam group, which affects the generation and migration of CO in the goaf, and easily leads to exceeding safety limits of CO in the return corner of the working face, which threatens the safety of underground production. To examine this problem, taking Lijiahao Coal Mine as an example, this study analyses the generation law of CO gas, the distribution law of overburden fractures, the characteristics of air leakage in the goaf, the sources of CO in the return corner, and the migration and accumulation law of CO in the goaf under multi-source air leakage in the mining of shallow buried close distance coal seam group through experiment tests, numerical simulations, observations and theoretical analyses. The results indicated that there is an exponential growth relationship between the CO generation rate and the coal temperature, and the critical temperature for rapid oxidation of coal samples is between 70 and 80 °C. The 31,115 working face has complicated air leakage from the working face and ground surface and the goaf of this coal seam. The surface air converges to the return corner through the mining fissure of overburden and 2-2 coal goaf, and the air leakage of the working face flows out from the return roadway through the goaf. The gas leakage in the overlying goaf and the oxidation of residual coal are the main sources of CO in the return corner. The CO generated during the coal mining process and the CO generated by the trackless rubber-tired vehicle operation will increase the CO concentration in the return corner to varying degrees. Under the effect of multi-source air leakage, CO from the overlying goaf and the residual coal in the goaf of this coal seam are migrated to the air return side of the goaf, resulting in the accumulation of CO in the return corner, and both of them have a linear positive correlation with the CO concentration in the return corner. The results of the study have scientific guidance for the control of air leakage and the prevention of CO excess in the goaf.

A new pressurization-insulation and pre-sealing system to improve pressure in cubic press from 6 GPa to 12 GPa.

In this paper, a pressurization-insulation and pre-sealing (PIPS) system is designed to increase the cell pressure of the widely used large volume cubic press without sacrificing cell volume. The sample chamber was sandwiched between a pair of tungsten carbide anvils used as the pressurization system. Ultra-high pressure in the cavity was up to about 12 GPa, and the pressure limit had increased by 100% in contrast with that of an anvil-gasket (AG) system. Furthermore, the confining pressure around the sample chamber was supported by grade 304 stainless steel and a zirconia-calcium oxide solid solution before a press load of 2.8 MN was applied as well as by four surrounding anvils. The relationship between the sample chamber pressure and the press load for this system was calibrated at room temperature using transitions in zinc telluride. With samples of similar volumes, the proposed system retained not only stability but also uniform pressure and temperature fields, in contrast with the AG system and the anvil-preformed gasket cubic press pressurization system. The results of more than 20 experiments show that the proposed PIPS system can operate stably under a press load of 4.2 MN, corresponding cell pressure of 10 GPa, and temperature in the cell exceeding 1800 °C by using graphite as a heater.

Transcriptomic analysis of the salivary gland of medicinal leech Hirudo nipponia.

Hirudo nipponia (known as Shui Zhi in Chinese) is a well-known Chinese medicine with numerous active ingredients in its body, especially in its saliva. This native Chinese blood-sucking leech has been used for therapeutic purposes since before 100 AD. Modern Chinese physicians use it for a wide range of diseases. Genomic data and molecular information about the pharmacologically active substances produced by this medicinal leech are presently unavailable despite this organism's medicinal importance. In this study, we performed transcriptome profiling of the salivary glands of medicinal leech H. nipponia using the Illumina platform. In total, 84,657,362 clean reads were assembled into 50,535 unigenes. The obtained unigenes were compared to public databases. Furthermore, a unigene sequence similarity search and comparisons with the whole transcriptome of medical leech were performed to identify potential proteins. Finally, more than 21 genes were predicted to be involved in anticoagulatory, antithrombotic, antibacterial, anti-inflammatory and antitumor processes, which might play important roles in the treatment of various diseases. This study is the first analysis of a sialotranscriptome in H. nipponia. The transcriptome profile will shed light on its genetic background and provide a useful tool to deepen our understanding of the medical value of H. nipponia.

Culture conditions and nutrition requirements for the mycelial growth of Isaria farinosa (Hypocreales: Cordycipitaceae) and the altitude effect on its growth and metabolome.

Isaria farinosa is a pathogen of alpine Thitarodes larvae that are hosts for the Chinese medicinal fungus, Ophiocordyceps sinensis. A matrix analysis indicated that the optimal culture conditions for the mycelial growth of I. farinosa are a 50-mL liquid broth in a 250-mL flask at more than 100-rpm rotation and 15-25 °C. Illumination does not affect the mycelial growth. The optimal nutrition requirements are D-(+)-galactose and D-(-)-fructose as carbon resources and D-cysteine as well as yeast powder, peptone, and beef extract as nitrogen resources at a carbon-to-nitrogen ratio of 1:1 to 1:7. The mineral component and vitamins also significantly increase the mycelial growth of I. farinosa. Based on the optimal culture conditions and nutrition requirements for the mycelial growth of I. farinosa, the effects of altitude on mycelial growth and its metabolome were evaluated using quadrupole-time-of-flight/mass spectrometry, principal component analysis and partial least squares discriminant analysis. The altitude did not affect the mycelial production but significantly regulated its metabolome. The study presents a new approach to better select a method for producing more useful metabolites and highlights the necessity of establishing standards for culturing methods related to altitude to preserve fungal quality; additionally, the results indicate that the use of a fermenter may meet the demands of large-scale mycelial production.

Complete mitochondrial genome of Hepialus xiaojinensis (Lepidoptera: Hepialidae).

Although Hepialus xiaojinensis is important as the host of Ophiocordyceps sinensis, it remains poorly known especially in genetic structure and phylogeny status. To get a better understanding of it, the complete mitochondrial genome sequence of H. xiaojinensis was sequenced for the first time. The genome was 15 397 bp in size and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and an AT-rich region, and the gene composition and the arrangement of which were consistent with other insects of Hepialidae. The base composition of the genome was A (41.44%), T (39.68%), C (11.52%), and G (7.36%), with an AT content of 81.13%. Moreover, we have performed phylogenetic analysis of H. xiaojinensis and other 11 Lepidoptera species.

Anti-herpes simplex virus activities of bioactive extracts from Antrodia camphorata mycelia.

BACKGROUND: Antrodia camphorata, a traditional Chinese medicine, is widely used in the treatment of liver diseases and cancers. Anti-inflammatory properties have also been described. HSV infection represents one of the most serious public health concerns globally because of its devastating impact. Searching for new antiviral agents, especially those with different mechanisms of action, is a crucial goal and there is an unmet need for alternative and complementary therapy against HSV infection. In this study, anti-herpes screening was performed with extracts from A. camphorata mycelia. METHODS: MTT assay, fractional inhibitory concentration index and median-effect principle were used to evaluate antiviral activity and to calculate drug combination effect. RESULTS: Crude ethanol extracts and isolated constituents showed inhibition of HSV replication at a very low concentration. Fraction A and antrodin A showed viral inhibitory effect with reduction of viral cell-to-cell spread. In addition, neither fraction A nor antrodin A showed interaction in combination with acyclovir. CONCLUSIONS: A. camphorata mycelia and antrodin A might have potential use as anti-HSV agents and are promising candidates for future antiviral drug design.

Complete mitochondrial genome of Hepialus gonggaensis (Lepidoptera: Hepialidae), the host insect of Ophiocordyceps sinensis.

The complete mitochondrial genome sequence of Hepialus gonggaensis was sequenced for the first time. The complete mtDNA sequence was 15,940 bp in length and contained 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and an AT-rich region, the gene composition and the arrangement of which were identical to other insects of Hepialidae. The overall base composition of the heavy strand was 41.14% A, 40.24% T, 11.17% C, and 7.45% G, with an AT content of 81.37%. The necleotide sequence data of 13 protein-coding genes of H. gonggaensis and other 10 Lepidoptera species were used for constructing the phylogenetic tree. It revealed that H. gonggaensis and other four Hepialidae species were clustered to a clade with high bootstraps values.

[Histological Study on Integumentary and Digestive System of Hirudo nipponica].

Objective: The integumentary and digestive system of Hirudo nipponica were studied,in order to protect the precious Chinese herbal medicine. Methods: The histological study of integumentary and digestive system of Hirudo nipponica by histological methods were carried out by microscopical method. Results: The body wall of Hirudo nipponica could be divided into five layers, including cuticular layer, epithelial layer, dermis layer, muscle layer and grape-like tissue. The digestive system was composed ofmouth,pharyngeal,esophagus,crop,intestine,rectum and anus. Conclusion: This study can provide the basic data for the artificial breeding, research in pathology and physiological functions of Hirudo nipponica.

Review on Natural Enemies and Diseases in the Artificial Cultivation of Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

Ophiocordyceps sinensis (syn. Cordyceps sinensis), well known as DongChongXiaCao (DCXC), is one of the most valuable traditional Chinese medicinal species. In this article, we provide a systematic review of natural enemies and diseases encountered in artificial cultivation of DCXC. Unfortunately, DCXC has been endangered over the past decades due to overharvesting and a worsening ecological environment. Therefore, the artificial cultivation of DCXC has been extensively investigated in recent years. Complete indoor artificial cultivation and semi-field cultivation are the two most common strategies used to cultivate DCXC. However, cultured DCXCs are often attacked by various natural enemies and diseases, which have resulted in substantial loss of the valuable medicinal resource. In this study, we have summarized the species of natural enemies and types of diseases confronted by DCXC. Twenty reported natural enemy species are categorized into four classes, one of which is reported for the first time in this study. Moreover, six microbial pathogens are also discussed. The recapitulation of the natural enemies and diseases in DCXC artificial cultivation not only promote the development of integrated pest management of DCXC cultivation but also provide important information to help preserve and develop this valuable resource.

[Effect of wound to growth of larva of host to Ophiocordyceps sinensis during artificial breeding].

To clear the effect of the wound to the growth of the larva of the host to the Ophiocordyceps sinensis, the wounds of same severity at the same position were made artificially to the larva and which were artificial fed at the same environment and condition. The results indicated that, over the winter, the survival rate of the wounded of the infection larva was lower than that of the healthy larva, but the weight had no significant difference between the wounded and the healthy larva. The survival rate of the wounded of the no infection larva was lower than that of the healthy larva, but except with black skin, the wounded larva with offwhite and dusty red had no influence on the variety of the weight. In summery, wound had no advantage to the survival rate, but had no influence to the weight. The result had provided theoretical basis to the reforming of the system of the artificial culture O. sinensis.

[Study on Reproductive Performance of Hirudio nipponica].

OBJECTIVE: To investigate the reproductive performance of Hirudo nipponica under artificial cultivation, in order to provide reference for the commercial rearing and breeding industry of this species. METHODS: The mean number of cocoons, cocoon weight, hatching numbers of cocoons, and the effect of parent leeches weight and density on reproductive performance were investigated. RESULTS: The mean number of cocoons produced by parent leeches was 0.9000 ± 0.5525, the range was from 0 to 2. The hatching number of cocoons were between 3 and 21, and the weight of cocoons were from 0.05 to 0.25 g. Length of newly hatched juvenile was 1.21 ± 0.22 cm, and the range was from 0.82 to 1.55 cm. It was found that the parent leeches weight, density and the cocoons weight were positively correlated to the reproductive performance of Hirudio nipponica. CONCLUSION: In artificial cultivation, moderate weight, age and density of parent leeches is necessary to be chose for increasing the reproductive performance of Hirudio nipponica.

Molecular Cloning and Characterization of a Subtilisin-Like Serine Protease Gene (Pr1) from the Medicinal Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes).

In the Chinese caterpillar mushroom Ophiocordyceps sinensis, a subtislin-like serine protease (Pr1) is one of the most important enzymes for its infection activity against insect cuticles. The Pr1 gene was isolated from the valuable Chinese medicinal fungus O. sinensis using rapid amplification of 5' and 3' complementary DNA ends. The 2079-bp full-length complementary DNA sequence containing the 1605-bp predicted open reading frame of the Pr1 gene was obtained (GenBank accession no. KF836756). The open reading frame encodes a protein comprising 534 amino acids. Protein sequence multiple alignment analysis revealed high homology with 16 other subtilisin serine proteases and exhibited the highly conserved catalytic domain (D195, H227, and S393). We also constructed a phylogenetic tree in this study. Further molecular studies are needed to elucidate the mechanisms of fungal infection.

[Expression of subtilisin-like protease gene from Hirsutella sinensis before and after infecting Hepialus gonggaensis].

To address the role of Pr1 gene in the process of Hirsutella sinensis infecting Hepialus gonggaensis, differential expression of subtilisin-like protease gene was detected. In the present study, Pr1 gene analogues from H. sinensis were obtained by PCR strategy using specific primers designed from conserved regions of Pr1 gene reported in the GenBank. Then we detected the changes in the expression of Pr1 gene before and after infecting H. gonggaensis using real-time quantitative PCR. We obtained the partial sequence of Pr1 gene with the length of 535 bp (GenBank accession: KC009680). Real-time PCR results showed that the expression level of Pr1 gene was significantly different among 8 samples (P < 0.01). Pr1 gene showed the obvious higher expression level (2-3 folds) after infecting the H. gonggaensis, suggesting that the Pr1 gene may play an important role in the process of H. sinensis infecting H. gonggaensis. The present study paves a way for further identification on infectivity assessment of H. sinensis.

[Relevance analysis of amino acid contents in cultivated Cordyceps and cultivation materials].

OBJECTIVE: To clarify the relevance of amino acid content in cultivated Cordyceps and cultivation materials. METHOD: The content of amino acid was determined with L-8800 amino acid analyzer, and the relevance of amino acid content was analyzed with SPSS. RESULT AND CONCLUSION: Except mycelium of the C. sinensis or the blood-lymph of the larva, the cultivated Cordyceps and the main relevant cultivation materials had detected to contain all kinds of amino acids. Except among the mycelium, the blood-lymph of the larva, the part of the larva or of the stroma of cultivated Cordyceps, there was distinct relevance of amino acid contents in cultivated Cordyceps and the cultivation materials (P<0.01).

[Study on biological character of Hepialus introduced from Yunnan province].

OBJECTIVE: To study the biological character of Hepialus introduced from Yunnan province. METHOD: H. jianchuanensis introduced from Yunnan was bred artificially to observe its biological character. RESULT: The pupa duration was about 40 d, pupa emergence lasted for one day, female to male ratio was about 1.2:1, the mating peak was 19:30-20:30, the adults duration was about 25 d, the average life time of female was 5-6 d and that of male 6-7 d, the average number of the producing egg was about 300, the average weight of the 100 eggs was 10.33 mg. Lasting period of the larva is 350 d and the infancy larva showed the character of the polyphagy and cold endurance. CONCLUSION: Results indicate that there are some differences in the whole duration, weight of the 100 eggs and the number of the eggs left in the abdomen between H. jianchuanensis and others bred at the Kangding base.

[Analysis of the bacterial diversity in intestines of Hepialus gonggaensis larvae].

OBJECTIVE: We investigated the intestinal microbial diversity in the larval gut of Hepialus gonggaensis, an economically important insect. METHODS: We used morphological, physiological, chemotaxonomic characteristics and 16S rRNA analysis method, and the molecular method of PCR-DGGE (denaturing gradient gel electrophoresis) analysis based on the sequence of 16S rRNA V3 region gene. RESULTS: By the traditional isolation method, 8 genera of bacteria were identified from 11 isolated bacterial populations. The dominant bacteria in intestine belonged to enterobacter. By 16S rRNA V3 region gene DGGE method, eleven distinct bands were obtained from 16S rDNA amplificons. The bands were purified, sequenced. The sequences aligned with GenBank database and showed that they were belonged to 8 different genera of bacteria. Phylogenetic analysis showed that the sequences of bacteria belonged to the Proteobacteria and Firmicutes. The most dominant bacteria group was Carnobacterium in the gut and Bacillus followed by it. The different patterns were observed in different instars larvae guts from DGGE profiles, which might be related to their physiological development stages. CONCLUSION: 8 genera were obtained from intestine of H. gonggaensis by traditional culturing method and 16S rDNA analysis method respectively, but the two groups were not exactly same, and the dominant group was different also. This suggested that a combination of molecular and traditional culturing methods can be used to analyze and monitor the diversity of intestinal microflora effectively, and that will give us more information of microorganism diversity.