Allelopathic effects of phenolic acid extracts on Morchella mushrooms, pathogenic fungus, and soil-dominant fungus uncover the mechanism of morel continuous cropping obstacle.

The prominent problem of continuous cropping obstacle has been frustrating the morel farming. To deepen the understanding on morel continuous cropping obstacle, the allelopathic effects of phenolic acid extracts from morel continuous cropping soils on growth and development of Morchella sextelata, M. eximia, M. importuna, pathogenic fungus Fusarium sp. and soil-dominant fungus Chaetomium sp. were investigated. These effects were expressed as response index (RI). Under actual content of phenolic acids (6.150 µg/g fresh mixed continuous cropping soil), the mycelial growth of all tested morel strains was inhibited (RI < 0), while the allelopathic effect of control phenolic acids (4.252 µg/g fresh mixed control soil) was between promotion and inhibition, which suggested that the phenolic acid extracts from morel continuous cropping soils may exhibit certain extent of autotoxicity for the existence of morel-specific allelochemicals. In addition, the aggravated pigmentation and reduced occurrence of sclerotium in three Morchella fungi at growth inhibitory concentrations of phenolic acids indicated the induction of morel strain aging. Meanwhile, most concentrations of phenolic acids showed stimulatory effects on sporulation of Fusarium sp. and Chaetomium sp. (RI > 0), manifesting the enrichment of soil-borne pathogenic fungi and dominance of certain fungal population in soil ecosystem. Collectively, the allelopathic effects of phenolic acid extracts play an instrumental role in morel continuous cropping obstacle. The study will be beneficial for healthy development of morel artificial cultivation.

Co-occurrence patterns among prokaryotes across an age gradient in pit mud of Chinese strong-flavor liquor.

It is widely believed that the quality and characteristics of Chinese strong-flavor liquor (CSFL) are closely related to the age of the pit mud; CSFL produced from older pit mud tastes better. This study aimed to investigate the alteration and interaction of prokaryotic communities across an age gradient in pit mud. Prokaryotic microbes in different-aged pit mud (1, 6, and 10 years old) were analyzed by Illumina MiSeq sequencing of the 16S rRNA gene. Analysis of the 16S rRNA gene indicated that the prokaryotic community was significantly altered with pit mud age. There was a significant increase in the genera Methanosarcina, Methanobacterium, and Aminobacterium with increased age of pit mud, while the genus Lactobacillus showed a significant decreasing trend. Network analysis demonstrated that both synergetic co-occurrence and niche competition were dominated by 68 prokaryotic genera. These genera formed 10 hubs of co-occurrence patterns, mainly under the phyla Firmicutes, Euryarchaeota, and Bacteroidetes, playing important roles on ecosystem stability of the pit mud. Environmental variables (pH, NH4+, available P, available K, and Ca2+) correlated significantly with prokaryotic community assembly. The interaction of prokaryotic communities in the pit mud ecosystem and the relationship among prokaryotic communities and environmental factors contribute to the higher quality of the pit mud in older fermentation pits.

Interspecific hybridization between cultivated morels Morchella importuna and Morchella sextelata by PEG-induced double inactivated protoplast fusion.

The commercial production of Morchella mushrooms calls for urgent breeding of excellent varieties or strains with appropriate tools, such as protoplast fusion. However, the protoplast fusion in morels has not been studied. In this paper, interspecific hybridization between cultivated morels of M. importuna and M. sextelata by PEG-induced protoplast fusion was conducted. Apart from functional complementation of double inactivated protoplasts, the fusants were characterized by cultural and cultivated characters and molecular markers of random amplified polymorphic DNA (RAPD). The results suggested that the hybrids and their parents showed significant difference in their inoculum recovery time, mycelial growth rate, yield of cultivation and total amino acid content of ascocarps. Moreover, positive barrage reactions were observed between parental strains as well as between each parent and a hybrid line. A dendrogram created on the basis of RAPD fingerprints exhibited three major clusters, in which morel hybrids showed intra-cluster variations, M. sextelata #6 formed an out group, while M. importuna #4 was phylogenetically closer to morel hybrids. All the results demonstrated that real fusants were obtained in our study. Protoplast fusion may provide an ideal alternative for new strain selection, and thus will promote the healthy development of morel industry.

Hybrid mesoporous silica nanospheres modified by poly (NIPAM-co-AA) for drug delivery.

The synthesis of drug delivery systems based on surface-modified mesoporous silica hollow structures remains a huge challenge. In this paper, we have obtained hollow mesoporous silica nanoparticles (MSNs) by surfactant directed sol-gel assisted hydrothermal treatment. The MSNs have the inorganic-organic hybrid frameworks with uniform diameter distribution (260 nm), and their specific surface area, mesoporous size and pore volume are 540 m2 g-1, 3.7 nm, 0.58 cm3 g-1, respectively. It was proved that the preparation of hollow ethane-bridged nanospheres with two silicon source was due to the high crosslinking of the silicone interface and hydrothermal treatment, providing a new approach for the study of drug-loaded and controlled release behavior. Based on the synthesis of MSNs, MSNs were modified by methacryloxy propyl trimethoxyl silane (MPS) on the surface of MSNs. Then N-isopropylacryamide (NIPAM) and acrylic acid (AA) were grafted onto the surface of modified MSNs. The hollow ethane-bridged PNA-MSNs (poly (NIPAM-co-acrylic acid)-MSNs) with two silicon source were prepared successfully. Due to their distinctive hollow structure, PNA-MSNs demonstrated high drug encapsulation efficiency (70.4% ± 2.9%). The experiment results proved that the modified hollow nanoparticles not only had good biocompatibility and stability, but also possessed pH-/thermal-dual responsiveness in drug release.

Hybridization of graphene oxide into nanogels to acquire higher photothermal effects for therapeutic delivery.

Although the special architecture of two-dimensional (2D) nanomaterials endows them with unique properties, their poor colloidal stability remains a main bottleneck to fully exploit their applications in the biomedical field. Herein, this study aims to develop a simple and effective approach to in situ incorporate 2D graphene oxide (GO) nanoplatelets into a thermosensitive matrix to acquire hybrid nanogels with good stability and photothermal effect. In order to improve its stability, GO firstly underwent silanization to its surface with double bonds, followed by intercalation with N-isopropylacrylamide (NIPAM) in the presence of a disulfide-containing crosslinker via an emulsion method. Radical polymerization was then initiated to accelerate direct GO exfoliation in PNIPAM nanogels by forming covalent bonds between them. The well-dispersed GO nanopletlets in the nanogels not only displayed an enhanced photothermal effect, but also improved the encapsulation efficiency of an anticancer drug. The hybrid nanogels accelerate drug release under conditions mimicking the acidic/reducible solid tumor and intracellular microenvironments, most importantly, it can be further enhanced via remote photothermal treatment. The multifunctional nanogels potentiate their synergistic anticancer bioactivity as an ideal nanoplatform for cancer treatment.

Effect of carbon source on production, characterization and bioactivity of exopolysaccharide produced by Phellinus vaninii Ljup.

The effect on different three carbon source (i.e. glucose, fructose and sucrose) on production, chemical characterization and antioxidant activity of exopolysaccharide (EPS) produced by Phellinus vaninii Ljup was investigated in this study. Amongst carbon sources examined, glucose and sucrose were favorable for the mycelia growth, while the maximum EPS yield was achieved when sucrose was employed. The predominant carbohydrate compositions in EPSs identified were gluconic acid, glucose, mannose and galactose acid. Then, FT-IR spectral analysis revealed prominent characteristic groups in EPSs. EPSs molecule exist as nearly globular shape form in aqueous solution. The variation also affects antioxidant activities by investigated by using hydroxyl and DPPH radical scavenging assay. Sucrose was best carbon source from the viewpoint of antioxidant activity due to the relatively high contents of galactose in the EPS with moderate molecular weight and polydispersity.

Effect of Tween 80 and Acetone on the Secretion, Structure and Antioxidant Activities of Exopolysaccharides from Lentinus tigrinus.

In this study, the effects of the addition of Tween 80 and acetone on secretion, structure and antioxidant activities of Lentinus tigrinus exopolysaccharides (EPS) were investigated. It was found that Tween 80 and acetone displayed a stimulatory effect on EPS secretion. The EPS obtained by the addition of Tween 80 (EPS-T), acetone (EPS-A) and control (EPS--C) were purified by Sepharose CL-6B gel filtration chromatography and molecular mass of purified fractions was estimated to be 22.1, 137 and 12 kDa, respectively. Monosaccharide composition analysis indicated that EPS-T, EPS-A and EPS-C were mainly composed of glucose and mannose. Congo Red test indicated that EPS-T and EPS-A had a highly ordered conformation of triple helix, while EPS-C had a random coil conformation. Furthermore, EPS-A exhibited higher DPPH scavenging and antiproliferative activities than EPS--C and EPS-T, which might be attributed to the molecular mass.

[WANG Haidong's experience in treatment of wrist rheumatoid arthritis with acupotomy mobilization at the muscle regions of three yang meridians of hand].

The paper introduces professor WANG Haidong's clinical experience in treatment of wrist rheumatoid arthritis with acupotomy mobilization at the muscle regions (sinews/fascia) of three yang meridians of hand. Professor WANG Haidong believes that wrist rheumatoid arthritis belongs to the disorder of meridian muscle regions and is especially associated with the damage of the muscle regions of three yang meridians of hand running through the wrist. Under the guidance of meridian muscle region theory, on the basis of modern anatomy, and the treatment principle, "needling the affected areas may treat disorders of sinews/fascia and dysfunction of meridians simultaneously", acupotomy mobilization is adopted to balance sinews/fascia and bones, operated directly at the involved meridian muscle regions. Besides the foci (palpable knotted sites) on the distribution of muscle regions, acupoints along the affected meridians are stimulated in combination. With this therapy, after determining the location of illness, both the disorder of sinews/fascia and that of meridians can be treated.

Production, preliminary characterization, and bioactivities of exopolysaccharides from Pleurotus geesteranus 5(#).

The optimal culture conditions of exopolysaccharides (EPS) production in submerged culture medium by Pleurotus geesteranus 5(#) were determined using an orthogonal matrix method. The optimal defined medium (per liter) was 60.0 g maltose, 5.0 g tryptone, 1 mM NaCl, 5 mM KH(2)PO(4), and initial pH 6.0 at 28 °C. In the optimal culture medium, the maximum EPS production was 16.97 g/L in a shake flask. Two groups of EPSs (designated as Fr-I and Fr-II) were obtained from the culture filtrates by size exclusion chromatography (SEC), and their molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index (RI) detector system. The approximate weight-average molar masses of the Fr-I and Fr-II of EPS were determined to be 3.263 × 10(4) and 5.738 × 10(3) g/mol, respectively. The low values of polydispersity ratio (1.176 and 1.124 for Fr-I and Fr-II, respectively) of EPSs mean that these EPS molecules exist much less dispersed in aqueous solution without forming large aggregates. Furthermore, the experiments in vitro indicated that P. geesteranus 5(#) EPS exhibit high antitumor and antioxidative effects.

Activity Assay of Amylase and Xylanase Is Available for Quantitative Assessment of Strain Aging in Cultivated Culinary-Medicinal Morchella Mushrooms (Ascomycotina).

Strain aging has been mainly contributing to the "uncertainty" of Morchella farming. The situation calls for urgent quantitative assessment of strain aging in cultivated Morchella mushrooms. In this paper, systemic senescence of the productive strains of M. eximia, M. importuna, and M. sextelata was achieved through successive subculturing to provide subcultures with different degree of aging for further studies. Then the quantitative assessment of morel strain aging was conducted by activity assay of amylase and xylanase using dinitrosalicylic acid (DNS) method. The results suggested that both activity of amylase and xylanase decreased along with the rise of subculture times. Meanwhile, the correlation between xylanase activity and time of subculturing in the tested morel strains was higher than that of amylase assay. Consequently, assay of amylase and xylanase activity by DNS method can be used in the quantitative assessment of morel strain aging, and assay of xylanase activity is the better alternative. The work will improve the settlement of "uncertainty" in the morel industry and thus be beneficial for stable development of morel farming.

Large-Scale Field Cultivation of Morchella and Relevance of Basic Knowledge for Its Steady Production.

Morels are one of the most highly prized edible and medicinal mushrooms worldwide. Therefore, historically, there has been a large international interest in their cultivation. Numerous ecological, physiological, genetic, taxonomic, and mycochemical studies have been previously developed. At the beginning of this century, China finally achieved artificial cultivation and started a high-scale commercial development in 2012. Due to its international interest, its cultivation scale and area expanded rapidly in this country. However, along with the massive industrial scale, a number of challenges, including the maintenance of steady economic profits, arise. In order to contribute to the solution of these challenges, formal research studying selection, species recognition, strain aging, mating type structure, life cycle, nutrient metabolism, growth and development, and multi-omics has recently been boosted. This paper focuses on discussing current morel cultivation technologies, the industrial status of cultivation in China, and the relevance of basic biological research, including, e.g., the study of strain characteristics, species breeding, mating type structure, and microbial interactions. The main challenges related to the morel cultivation industry on a large scale are also analyzed. It is expected that this review will promote a steady global development of the morel industry based on permanent and robust basic scientific knowledge.

Ultrastructure and Physiological Characterization of Morchella Mitospores and Their Relevance in the Understanding of the Morel Life Cycle.

Morels, which belong to the Ascomycete genus Morchella, are highly valued edible fungi treasured by gourmet chefs worldwide. Some species are saprotrophic and others are able to form facultative mycorrhizal-like associations with plant roots without establishing true ectomycorrhizal symbioses. In general, it is considered that the formation of asexual spores, or mitospores, is an important step in the life cycle of morels. However, ultrastructure characterization and physiological attributes of morel mitospores have received little attention. In this contribution, the mitospores of M. sextelata were successfully induced under laboratory conditions and their ultrastructure, occurrence, germination, physiological characteristics and mating type gene structure were studied. Mitospore production was closely related to aeration, nutrition and humidity conditions. The average germination rate of mitospores on different media and under various induction stimuli was very low, with an average of 1/100,000. Based on the ultrastructure characterization, low germination rate, growth rate decline, rapid aging and mating genotyping, it was concluded that the mitospores of M. sextelata had lost their conventional function as conidia and might act more as mate sperm-like (gamete) structures. Thus, this study contributed to a deeper understanding of the life cycle of the economically and ecologically important morel fungal group.

Structure of the Mating-Type Genes and Mating Systems of Verpa bohemica and Verpa conica (Ascomycota, Pezizomycotina).

Verpa spp. are potentially important economic fungi within Morchellaceae. However, fundamental research on their mating systems, the key aspects of their life cycle, remains scarce. Fungal sexual reproduction is chiefly governed by mating-type genes, where the configuration of these genes plays a pivotal role in facilitating the reproductive process. For this study, de novo assembly methodologies based on genomic data from Verpa spp. were employed to extract precise information on the mating-type genes, which were then precisely identified in silico and by amplifying their single-ascospore populations using MAT-specific primers. The results suggest that the MAT loci of the three tested strains of V. bohemica encompassed both the MAT1-1-1 and MAT1-2-1 genes, implying homothallism. On the other hand, amongst the three V. conica isolates, only the MAT1-1-1 or MAT1-2-1 genes were present in their MAT loci, suggesting that V. conica is heterothallic. Moreover, bioinformatic analysis reveals that the three tested V. bohemica strains and one V. conica No. 21110 strain include a MAT1-1-10 gene in their MAT loci, while the other two V. conica strains contained MAT1-1-11, exhibiting high amino acid identities with those from corresponding Morchella species. In addition, MEME analysis shows that a total of 17 conserved protein motifs are present among the MAT1-1-10 encoded protein, while the MAT1-1-11 protein contained 10. Finally, the mating type genes were successfully amplified in corresponding single-ascospore populations of V. bohemica and V. conica, further confirming their life-cycle type. This is the first report on the mating-type genes and mating systems of Verpa spp., and the presented results are expected to benefit further exploitation of these potentially important economic fungi.

Production, characterization and antioxidant activity of exopolysaccharides from submerged culture of Morchella crassipes.

The aim of this work was to investigate the fermentation optimization, molecular characterization, and antioxidant activity in vitro of exopolysaccharides (EPS) from Morchella crassipes in submerged culture. Firstly, an optimal medium for EPS production was obtained by single-factor experiment and central composite design as follows: maltose 44.79 g/L and tryptone 4.21 g/L. Then, one fraction of EPS was obtained from the culture filtrates by size exclusion chromatography and the molecular characteristics were examined by a multi-angle laser light scattering and refractive index detector system. The weight-average molar mass and the polydispersity ratio of the EPS fraction were revealed to be 1.961 × 10(4) g/mol and 1.838, respectively. FT-IR spectroscopy was used for obtaining vibrational spectra of the purified EPS fraction. Finally, the antioxidant activity of EPS was investigated and the relationship with molecular properties was discussed as well.

Production, purfication, molecular characterization and bioactivities of exopolysaccharides produced by the wine cap culinary-medicinal mushroom, Stropharia rugosoannulata 2# (higher Basidiomycetes).

The optimal culture conditions of exopolysaccharides (EPS) production in submerged culture medium by Stropharia rugosoannulata 2# were determined using the orthogonal matrix method. The optimal defined medium (per liter) was 60.0 g sucrose, 6.0 g tryptone, 5 mM KH2PO4, and initial pH 7.0 at 28°C. In the optimal culture medium, the maximum EPS production was 9.967 g/L in shake-flask culture. One fraction of EPS was purified from the culture filtrates by size exclusion chromatography (SEC), and the molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index (RI) detector system. The weight-average molar masses and the polydispersity ratio of the EPS fraction were determined to be 5.305 × 103 g/mol and 2.014, respectively. FTIR spectroscopy was used for obtaining vibrational spectra of the purified EPS fraction. The obvious characteristic absorption at 884.3 cm-1 revealed the existence of ß configuration. Furthermore, the experiments in vitro indicated that S. rugosoannulata 2# EPS exhibit high antitumor and antioxidative effects.

The prokaryotic community, physicochemical properties and flavors dynamics and their correlations in fermented grains for Chinese strong-flavor Baijiu production.

Fermented grain (FG), a complex and unique ecosystem, is the main microbial habitats, biochemical reaction system and direct source of flavor compounds for the Chinese strong-flavor Baijiu (CSFB) production. However, the dynamics of physicochemical properties, prokaryotic community and flavor compounds of FGs during the long-term fermentation process are still not completely clear. Here, the above topics on FGs in the actual production process were comprehensively studied by using a combination of physicochemical analysis, GC-MS detection and Illumina HiSeq sequencing methods. The whole fermentation process could be divided into two stages including early (0-25d) and the later stage (25-60d) based on the dynamics of FG physicochemical properties and the changes of prokaryotic community diversity. A total of 41phyla and 364 genera were detected, and 9 of them were dominant genera in FG complex ecosystem, including Lactobacillus, Pediococcus, Ochrobactrum, Bacillus etc. Among them, the dynamics of 29 top10 genera in FGs were mainly influenced by the starch and total acid, followed by NH4+ and ethanol, and 7 genera (hubs, e.g., Clostridium, Methanosaeta, Bacillus, etc.) of them may play important roles in FG ecosystem stability. A total of 71 volatiles including 33 esters, 14 alcohols, 9 fatty acids, 5 phenols, and 10 other compounds were detected in the FGs, and most of them formed in the early stage. Some important flavor substances (e.g., ethyl octanoate, 3-methylbutanol, hexanoate, etc.) increased in the later stage. Moreover, the formation of some flavor compound might require multiple microbes involved. For instance, ten of the top10 genera, including Lactobacillus, Clostridium, Methanosarcina, Sedimentibacter, Bacillus, etc., were significantly and positively correlated with four important esters. This study may help to clarify the complex correlations among prokaryotic community, physicochemical properties and flavors, allow the improvement of CSFB quality by using bioaugmentation and/or controlling environmental factors, and shed more light on the ecological rules guiding community assembly in FGs.

The thermal/pH-sensitive drug delivery system encapsulated by PAA based on hollow hybrid nanospheres with two silicon source.

The synthesis of drug delivery systems based on hollow mesoporous silica nanoparticles (MSNs) is still a major challenge. In this work, the hollow hybrid MSNs were successfully prepared by cetyltrimethylammonium bromide-directed sol-gel process and one-step hydrothermal treatment process. The hollow hybrid MSNs had hybrid ethane-bridged frameworks with the uniform particle size (250 nm) and mesoporous pore diameter (3.7 nm). The polyacrylic acid (PAA) encapsulated drug delivery system based on hollow hybrid MSNs was prepared by using silanization, surface modification, doxorubicin hydrochloride (DOX) loading, and PAA coating. Drug encapsulation and release behavior at different temperatures and pH were studied by using DOX as a model guest molecule. The results displayed that the modified hollow ethane-bridged MSNs possessed good biocompatibility and excellent thermal/pH-dual-sensitive drug release property. This novel thermal/pH-sensitive drug delivery system based on hollow ethane-bridged MSNs has the advantages of feasible synthesis, no cytotoxicity, and good drug loading capacity, which may have potential applications in the anticancer therapy.

Discovery and identification of medium-chain fatty acid responsive promoters in Saccharomyces cerevisiae.

Medium-chain fatty acids (MCFAs) and their derivatives are important chemicals that can be used in lubricants, detergents, and cosmetics. MCFAs can be produced in several microbes, although production is not high. Dynamic regulation by synthetic biology is a good method of improving production of chemicals that avoids toxic intermediates, but chemical-responsive promoters are required. Several MCFA sensors or promoters have been reported in Saccharomyces cerevisiae. In this study, by using transcriptomic analysis of S. cerevisiae exposed to fatty acids with 6-, 12-, and 16-carbon chains, we identified 58 candidate genes that may be responsive to MCFAs. Using a fluorescence-based screening method, we identified MCFA-responsive promoters, four that upregulated gene expression, and three that downregulated gene expression. Dose-response analysis revealed that some of the promoters were sensitive to fatty acid concentrations as low as 0.02-0.06 mM. The MCFA-responsive promoters reported in this study could be used in dynamic regulation of fatty acids and fatty acid-derived products in S. cerevisiae.

Optimizing the DO-stat protocol for enhanced production of thermostable pullulanase in Escherichia coli by using oxygen control strategies.

Production of a thermostable pullulanase by DO-stat fed-batch fermentation of recombinant Escherichia coli BL 21 was investigated in a 5 L of fermentor. The effect of three oxygen control strategies, glucose feedback, shifting fermentor pressure, and adding oxygen-enriched air, on cell growth and pullulanase expression were examined. The oxygen-transfer capacity was found to be enhanced with increasing fermentor pressure and oxygen ratio in oxygen-enriched air, but the cell growth and pullulanase production were restrained under high fermentor pressure. The highest cell density and pullulanase activity reached 55.1 g/L and 412 U/mL, respectively, in the case by adding oxygen-enriched air, which was suggested as an effective approach to enhance both cell growth and pullulanase production. PRACTICAL APPLICATIONS: This thermostable pullulanase displayed optimal activity at 90°C and pH 5.4, which could be applied for one-step saccharification of starch biomass. The optimization of the DO-stat fed-batch fermentation in high cell density level would provide a research basis for its industrialization.

Tumor-mediated shape-transformable nanogels with pH/redox/enzymatic-sensitivity for anticancer therapy.

Lack of sufficient tumor penetration of the current nanomedicines is a major reason limiting their clinical success in cancer therapy. In this work, we aimed at the development of a novel biodegradable nanoplatform for the selective and controlled delivery of anticancer agents, with improved tumor permeability and the ability to release ultrasmall nanovesicles in the tumor microenvironment. To this end, positively charged nanogels were obtained through the double-crosslinking of chitosan with an ionic physical gelator and a disulfide-containing chemical crosslinker. After conjugation to an anionic oligomer, the cationic nanogels were transformed into negatively charged nanocarriers (CTCP), enabling effective encapsulation of the cationic anticancer agent doxorubicin (DOX) to generate a biodegradable nanomedicine (DOX@CTCP). DOX@CTCP could maintain sustained DOX release and decreased DOX toxicity. Upon arrival at the tumor tissue, the reductive and lysozyme-high microenvironment drives the cleavage of the nanomedicine to release DOX-carrying nanoblocks of smaller size, which together with their acidic-protonable feature achieves an effective therapeutic delivery into cancer cells. The nanomedicine described here showed excellent biocompatibility/biosafety and enhanced in vivo antitumor efficacy.