Functional Guilds, Community Assembly, and Co-occurrence Patterns of Fungi in Metalliferous Mine Tailings Ponds in Mainland China.

Metalliferous mine tailings ponds are generally characterized by low levels of nutrient elements, sustained acidic conditions, and high contents of toxic metals. They represent one kind of extreme environments that are believed to resemble the Earth's early environmental conditions. There is increasing evidence that the diversity of fungi inhabiting mine tailings ponds is much higher than previously thought. However, little is known about functional guilds, community assembly, and co-occurrence patterns of fungi in such habitats. As a first attempt to address this critical knowledge gap, we employed high-throughput sequencing to characterize fungal communities in 33 mine tailings ponds distributed across 18 provinces of mainland China. A total of 5842 fungal phylotypes were identified, with saprotrophic fungi being the major functional guild. The predictors of fungal diversity in whole community and sub-communities differed considerably. Community assembly of the whole fungal community and individual functional guilds were primarily governed by stochastic processes. Total soil nitrogen and total phosphorus mediated the balance between stochastic and deterministic processes of the fungal community assembly. Co-occurrence network analysis uncovered a high modularity of the whole fungal community. The observed main modules largely consisted of saprotrophic fungi as well as various phylotypes that could not be assigned to known functional guilds. The richness of core fungal phylotypes, occupying vital positions in co-occurrence network, was positively correlated with edaphic properties such as soil enzyme activity. This indicates the important roles of core fungal phylotypes in soil organic matter decomposition and nutrient cycling. These findings improve our understanding of fungal ecology of extreme environments.

Remarkable effects of microbial factors on soil phosphorus bioavailability: A country-scale study.

Low soil phosphorus (P) bioavailability causes the widespread occurrence of P-limited terrestrial ecosystems around the globe. Exploring the factors influencing soil P bioavailability at large spatial scales is critical for managing these ecosystems. However, previous studies have mostly focused on abiotic factors. In this study, we explored the effects of microbial factors on soil P bioavailability of terrestrial ecosystems using a country-scale sampling effort. Our results showed that soil microbial biomass carbon (MBC) and acid phosphatase were important predictors of soil P bioavailability of agro- and natural ecosystems across China although they appeared less important than total soil P. The two microbial factors had a positive effect on soil P bioavailability of both ecosystem types and were able to mediate the effects of several abiotic factors (e.g., mean annual temperature). Meanwhile, we revealed that soil phytase could affect soil P bioavailability at the country scale via ways similar to those of soil MBC and acid phosphatase, a pattern being more pronounced in agroecosystems than in natural ecosystems. Moreover, we obtained evidence for the positive effects of microbial genes encoding these enzymes on soil P bioavailability at the country scale although their effect sizes varied between the two ecosystem types. Taken together, this study demonstrated the remarkable effects of microbial factors on soil P bioavailability at a large spatial scale, highlighting the importance to consider microbial factors in managing the widespread P-limited terrestrial ecosystems.

Hidden diversity and potential ecological function of phosphorus acquisition genes in widespread terrestrial bacteriophages.

Phosphorus (P) limitation of ecosystem processes is widespread in terrestrial habitats. While a few auxiliary metabolic genes (AMGs) in bacteriophages from aquatic habitats are reported to have the potential to enhance P-acquisition ability of their hosts, little is known about the diversity and potential ecological function of P-acquisition genes encoded by terrestrial bacteriophages. Here, we analyze 333 soil metagenomes from five terrestrial habitat types across China and identify 75 viral operational taxonomic units (vOTUs) that encode 105 P-acquisition AMGs. These AMGs span 17 distinct functional genes involved in four primary processes of microbial P-acquisition. Among them, over 60% (11/17) have not been reported previously. We experimentally verify in-vitro enzymatic activities of two pyrophosphatases and one alkaline phosphatase encoded by P-acquisition vOTUs. Thirty-six percent of the 75 P-acquisition vOTUs are detectable in a published global topsoil metagenome dataset. Further analyses reveal that, under certain circumstances, the identified P-acquisition AMGs have a greater influence on soil P availability and are more dominant in soil metatranscriptomes than their corresponding bacterial genes. Overall, our results reinforce the necessity of incorporating viral contributions into biogeochemical P cycling.

[Preparation technology of total coumarins from Laportea bulbifera].

OBJECTIVE: To investigate the preparation process of total coumarins from Laportea bulbifera. METHODS: The optimum condition for the extraction of total coumarins was studied using the orthogonal test, and the extracts was purled with different kinds of macroporous resins. The content of total coumarins was detected by ultraviolet spectrophotometry. RESULTS: The optimum extraction process was as follows: three-time circumfluence with 4 times volume of 75% ethanol for 1.5 h each time. HPD300 macroporous resin was the optimum one for the separation and purification of total coumarins. The extraction condition was loading 30 mL of 0.4 g/mL the crude drug, flowing at 2BV/h, then edulcorating with 4 times volume of distilled water and eluting with the same volume of 75% ethanol. The yield rate was more than 52%. CONCLUSION: This method is suitable for the preparation of total coumarins in Laportea bulbifera, and the process is stable and repeatable.

Diverse Methylmercury (MeHg) Producers and Degraders Inhabit Acid Mine Drainage Sediments, but Few Taxa Correlate with MeHg Accumulation.

Methylmercury (MeHg) is a notorious neurotoxin, and its production and degradation in the environment are mainly driven by microorganisms. A variety of microbial MeHg producers carrying the gene pair hgcAB and degraders carrying the merB gene have been separately reported in recent studies. However, surprisingly little attention has been paid to the simultaneous investigation of the diversities of microbial MeHg producers and degraders in a given habitat, and no studies have been performed to explore to what extent these two contrasting microbial groups correlate with MeHg accumulation in the habitat of interest. Here, we collected 86 acid mine drainage (AMD) sediments from an area spanning approximately 500,000 km2 in southern China and profiled the sediment-borne putative MeHg producers and degraders using genome-resolved metagenomics. 46 metagenome-assembled genomes (MAGs) containing hgcAB and 93 MAGs containing merB were obtained, including those from various taxa without previously known MeHg-metabolizing microorganisms. These diverse MeHg-metabolizing MAGs were formed largely via multiple independent horizontal gene transfer (HGT) events. The putative MeHg producers from Deltaproteobacteria and Firmicutes as well as MeHg degraders from Acidithiobacillia were closely correlated with MeHg accumulation in the sediments. Furthermore, these three taxa, in combination with two abiotic factors, explained over 60% of the variance in MeHg accumulation. Most of the members of these taxa were characterized by their metabolic potential for nitrogen fixation and copper tolerance. Overall, these findings improve our understanding of the ecology of MeHg-metabolizing microorganisms and likely have implications for the development of management strategies for the reduction of MeHg accumulation in the AMD sediments. IMPORTANCE Microorganisms are the main drivers of MeHg production and degradation in the environment. However, little attention has been paid to the simultaneous investigation of the diversities of microbial MeHg producers and degraders in a given habitat. We used genome-resolved metagenomics to reveal the vast phylogenetic and metabolic diversities of putative MeHg producers and degraders in AMD sediments. Our results show that the diversity of MeHg-metabolizing microorganisms (particularly MeHg degraders) in AMD sediments is much higher than was previously recognized. Via multiple linear regression analysis, we identified both microbial and abiotic factors affecting MeHg accumulation in AMD sediments. Despite their great diversity, only a few taxa of MeHg-metabolizing microorganisms were closely correlated with MeHg accumulation. This work underscores the importance of using genome-resolved metagenomics to survey MeHg-metabolizing microorganisms and provides a framework for the illumination of the microbial basis of MeHg accumulation via the characterization of physicochemical properties, MeHg-metabolizing microorganisms, and the correlations between them.

[Study on effect of total coumarins from Urtica dentata on dextran sulfate sodium-induced colitis in mice].

OBJECTIVE: To study the effect of total coumarins (TC) from Urtica dentata on dextran sulfate sodium (DSS)-induced colitis in mice. METHOD: The colitis model was established by administering DSS. Having been treated with TC, their body weight was determined. Concentrations of IL-6, IL-10, TGF-beta1 and IFN-gamma were monitored by ELISA. Colon samples were collected for the histopathological examination. Western blot was used to detect TLR4 and NF-kappaB protein expression in colonic tissues. RESULT: TCs from U. dentata effectively controlled the body weight loss of mice with colitis, down-regulated the concentration of IL-6 and IFN-gamma and increased the suppressive cytokines IL-10 and TGF-beta1 in the serum. Additionally, TC alleviated the mucosal damage and decreased the expressions of TLR4 and NF-kappaB in colonic tissues. CONCLUSION: TCs from U. dentata shows the anti-inflammatory effect on colitis in mice by reducing the expressions of TLR4 and NF-kappaB in colonic tissues and regulating pro-and anti-inflammatory cytokines.

Metagenomic and metatranscriptomic insights into sulfate-reducing bacteria in a revegetated acidic mine wasteland.

The widespread occurrence of sulfate-reducing microorganisms (SRMs) in temporarily oxic/hypoxic aquatic environments indicates an intriguing possibility that SRMs can prevail in constantly oxic/hypoxic terrestrial sulfate-rich environments. However, little attention has been given to this possibility, leading to an incomplete understanding of microorganisms driving the terrestrial part of the global sulfur (S) cycle. In this study, genome-centric metagenomics and metatranscriptomics were employed to explore the diversity, metabolic potential, and gene expression profile of SRMs in a revegetated acidic mine wasteland under constantly oxic/hypoxic conditions. We recovered 16 medium- to high-quality metagenome-assembled genomes (MAGs) containing reductive dsrAB. Among them, 12 and four MAGs belonged to Acidobacteria and Deltaproteobacteria, respectively, harboring three new SRM genera. Comparative genomic analysis based on seven high-quality MAGs (completeness >90% and contamination <10%; including six acidobacterial and one deltaproteobacterial) and genomes of three additional cultured model species showed that Acidobacteria-related SRMs had more genes encoding glycoside hydrolases, oxygen-tolerant hydrogenases, and cytochrome c oxidases than Deltaproteobacteria-related SRMs. The opposite pattern was observed for genes encoding superoxide reductases and thioredoxin peroxidases. Using VirSorter, viral genome sequences were found in five of the 16 MAGs and in all three cultured model species. These prophages encoded enzymes involved in glycoside hydrolysis and antioxidation in their hosts. Moreover, metatranscriptomic analysis revealed that 15 of the 16 SRMs reported here were active in situ. An acidobacterial MAG containing a prophage dominated the SRM transcripts, expressing a large number of genes involved in its response to oxidative stress and competition for organic matter.

Globally distributed mining-impacted environments are underexplored hotspots of multidrug resistance genes.

Mining is among the human activities with widest environmental impacts, and mining-impacted environments are characterized by high levels of metals that can co-select for antibiotic resistance genes (ARGs) in microorganisms. However, ARGs in mining-impacted environments are still poorly understood. Here, we conducted a comprehensive study of ARGs in such environments worldwide, taking advantage of 272 metagenomes generated from a global-scale data collection and two national sampling efforts in China. The average total abundance of the ARGs in globally distributed studied mine sites was 1572 times per gigabase, being rivaling that of urban sewage but much higher than that of freshwater sediments. Multidrug resistance genes accounted for 40% of the total ARG abundance, tended to co-occur with multimetal resistance genes, and were highly mobile (e.g. on average 16% occurring on plasmids). Among the 1848 high-quality metagenome-assembled genomes (MAGs), 85% carried at least one multidrug resistance gene plus one multimetal resistance gene. These high-quality ARG-carrying MAGs considerably expanded the phylogenetic diversity of ARG hosts, providing the first representatives of ARG-carrying MAGs for the Archaea domain and three bacterial phyla. Moreover, 54 high-quality ARG-carrying MAGs were identified as potential pathogens. Our findings suggest that mining-impacted environments worldwide are underexplored hotspots of multidrug resistance genes.

Intramuscular delivery of a naked DNA plasmid encoding proinsulin and pancreatic regenerating III protein ameliorates type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by inflammation of pancreatic islets and destruction of ß cells. Up to now, there is still no cure for this devastating disease and alternative approach should be developed. To explore a novel gene therapy strategy combining immunotherapy and ß cell regeneration, we constructed a non-viral plasmid encoding proinsulin (PI) and pancreatic regenerating (Reg) III protein (pReg/PI). Therapeutic potentials of this plasmid for T1DM were investigated. Intramuscular delivery of pReg/PI resulted in a significant reduction in hyperglycemia and diabetes incidence, with an increased insulin contents in the serum of T1DM mice model induced by STZ. Treatment with pReg/PI also restored the balance of Th1/Th2 cytokines and expanded CD4(+)CD25(+)Foxp3(+) T regulatory cells, which may attribute to the establishment of self-immune tolerance. Additionally, in comparison to the mice treated with empty vector pBudCE4.1 (pBud), attenuated insulitis and apoptosis achieved by inhibiting activation of NF-κB in the pancreas of pReg/PI treated mice were observed. In summary, these results indicate that intramuscular delivery of pReg/PI distinctly ameliorated STZ-induced T1DM by reconstructing the immunological self-tolerance and promoting the regeneration of ß cells, which might be served as a promising candidate for the gene therapy of T1DM.

A comprehensive synthesis unveils the mysteries of phosphate-solubilizing microbes.

Phosphate-solubilizing microbes (PSMs) drive the biogeochemical cycling of phosphorus (P) and hold promise for sustainable agriculture. However, their global distribution, overall diversity and application potential remain unknown. Here, we present the first synthesis of their biogeography, diversity and utility, employing data from 399 papers published between 1981 and 2017, the results of a nationwide field survey in China consisting of 367 soil samples, and a genetic analysis of 12986 genome-sequenced prokaryotic strains. We show that at continental to global scales, the population density of PSMs in environmental samples is correlated with total P rather than pH. Remarkably, positive relationships exist between the population density of soil PSMs and available P, nitrate-nitrogen and dissolved organic carbon in soil, reflecting functional couplings between PSMs and microbes driving biogeochemical cycles of nitrogen and carbon. More than 2704 strains affiliated with at least nine archaeal, 88 fungal and 336 bacterial species were reported as PSMs. Only 2.59% of these strains have been tested for their efficiencies in improving crop growth or yield under field conditions, providing evidence that PSMs are more likely to exert positive effects on wheat growing in alkaline P-deficient soils. Our systematic genetic analysis reveals five promising PSM genera deserving much more attention.

[Recombination of RegIII-proinsulin-pBudCE4.1 plasmid and its therapeutic effect on STZ-induced type 1 diabetes mellitus].

The aim of this study is to investigate the therapeutic effect of RegIII-proinsulin-pBudCE4.1 plasmid on streptozotocin (STZ)-induced type 1 diabetes mellitus and its underlying mechanisms. The model of type 1 diabetes mellitus was established by intraperitoneal injections of STZ (40 mg kg(-1)) to Balb/c mice for five consecutive days. Then, ten type 1 diabetic mice were intramuscularly injected with 100 microg RegIII-proinsulin-pBudCE4.1 plasmid for 4 weeks (one time/week) and the blood glucose levels were monitored every week; whereas another ten diabetic mice served as negative control group were injected with pBudCE4.1 vector at the same dose. Normal control and model control mice were treated with normal saline at identical volume under the same way. Western blotting, MTT assay, ELISA, HE staining and Tunel assay were applied to explore the underlying mechanisms. Results showed that RegIII-proinsulin-pBudCE4.1 plasmid ameliorated the hyperglycemia symptoms in diabetic mouse remarkably. It induced an immunological tolerance state in type 1 diabetic mice by inhibiting the proliferation of splenic lymphocytes and recovering Th1/Th2 balance evidenced by MTT and ELISA analysis. Furthermore, it elevated insulin concentration in the serum of type 1 diabetic mice and promoted the regeneration of beta cells supported by the results of HE staining and Tunel assay. In conclusion, RegIII-proinsulin-pBudCE4.1 plasmid possesses powerful anti-diabetic ability, which may be involved in the inducing of immunological tolerance and enhancing beta cells recovery.

[Joint application of 7 interventional pulmonology methods in early diagnosis of lung cancer].

OBJECTIVE: To evaluate the combination of 7 interventional pulmonology methods in early diagnosis of lung cancer. METHODS: A total of 467 patients with thoracic and pulmonary lesions (include hilum pulmonis lymphadenectasis, mediastinal lymphadenectasis, pulmonary scobination, lump, lamellar infiltration, small amount of pleural fluid and pleural scobination) had negative results via exfoliative cytology, bacteriology and routine bronchoscopy. All these patients had ultrathin bronchoscopy with biopsy and brushing. For those 155 cases whose foci were located at porta pulmonis, inner zone or median zone, the authors applied ultrathin bronchoscopy with biopsy and brushing guided by X-ray. For those 95 cases whose foci were located at median zone or outer zone and unconnected with chest wall, per cutem lung puncture needle aspiration was employed under the guidance of X-ray. For those 102 cases whose foci were tightly connected with pleural membrane, per cutem lung puncture biopsy was employed under the guidance of type-B ultrasonic. For those 59 cases with suspected central airway foci, auto-fluorescence bronchoscopic biopsy and brushing were employed. For those 67 cases with hilum pulmonis or mediastinal lymphadenectasis, endobronchial ultrasonic transbronchial needle aspiration (EBUS-TBNA) was employed. For those 23 cases with small amount of pleural fluid or pleural scobination, electronic thoracoscopic biopsy and brushing were employed. RESULTS: It was found that 118 cases were diagnosed by ultrathin bronchoscopic biopsy and brushing with a positive rate of 25.3% (118/467), 105 cases by ultrathin bronchoscopy with biopsy and brushing guided by X-ray with a positive rate of 67.7% (105/155), 63 cases by per cutem lung puncture needle aspiration under the guidance of X-ray with a positive rate of 66.3% (63/95), 69 cases by per cutem lung puncture biopsy under the guidance of type-B ultrasound with a positive rate of 67.6% (69/102), 18 cases by auto-fluorescence bronchoscopic biopsy and brushing with a positive rate of 35.3% (18/51), 52 cases by EBUS-TBNA with a positive rate of 77.6% (52/67), 12 cases by electronic thoracoscopic biopsy and brushing with a positive rate of 52.2% (12/23). The total positive diagnostic rate was 93.6% (437/467). And the diagnostic rate of < or = stage II lung cancer (3 cases carcinoma in situ, 84 stage I a, 63 stage Ib, 65 stage IIa and 44 stage IIb) was 82.7% (259/313). CONCLUSION: Joint application of these 7 interventional bronchoscopic techniques can significantly boost the rate of early diagnosis of lung cancer.

Construction of an oesophageal cancer-specific ceRNA network based on miRNA, lncRNA, and mRNA expression data.

AIM: To explore the expression profiles of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and mRNAs in oesophageal squamous cell carcinoma (ESCC) in order to construct an oesophageal cancer-specific competing endogenous RNA (ceRNA) network. METHODS: In this work, the expression data of miRNAs, lncRNAs, and mRNAs in ESCC were obtained. An oesophageal cancer-specific ceRNA network was then constructed and investigated. RESULTS: CeRNAs have the ability to reduce the targeting activity of miRNAs, leading to the de-repression of specific mRNAs with common miRNA response elements. CeRNA interactions have a critical effect in gene regulation and cancer development. CONCLUSION: This study suggests a novel perspective on potential oesophageal cancer mechanisms as well as novel pathways for modulating ceRNA networks for treating cancers.

Baicalin may alleviate inflammatory infiltration in dextran sodium sulfate-induced chronic ulcerative colitis via inhibiting IL-33 expression.

AIMS: To investigate the therapeutic effect of baicalin treatment in chronic ulcerative colitis (UC), and explore the potential anti-inflammation mechanism(s) via IL-33 pathway. MAIN METHODS: UC model were established by giving three cycles of 5-day 2% dextran sodium sulfate (DSS) with two intervals of 14-day recovery in mice, totaling 43days. At the 13th day of the UC modeling, mice received baicalin at doses of 50, 100, or 150mg/kg, respectively. Disease activity index (DAI) assessment as well as HE and PAS staining were performed. Serum levels of TNF-α, IL-1ß and IL-6 were determined by ELISA. Myeloperoxidase (MPO) activity and nitric oxide (NO) contents in colon were measured. The expressions of IL-33 and Ly6/G were examined by immunochemistry. And contents of IL-33 protein and NF-κB-related proteins were tested by Western blot. KEY FINDINGS: Morphological and histological analyses revealed that baicalin administration had a significant effect on reducing the severity of DSS-induced UC in mice. Besides, baicalin treatment significantly reduced the levels of MPO and NO. Moreover, increased levels of inflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, have been identified in damaged colon tissue, which was noticeably reduced by baicalin treatment. Our data demonstrated that protein levels of IL-33 and NF-κB p65 were elevated in colon tissues of chronic UC mice. Baicalin treatment significantly suppressed levels of IL-33 and NF-κB p65, whereas levels of IκB-α were increased. SIGNIFICANCE: Baicalin treatment effectively alleviated DSS-induced chronic UC, and the protective mechanisms may involve inhibition of IL-33 expression and subsequent NF-κB activation.

Therapeutic effects of total coumarins from Urtica dentata Hand on collagen-induced arthritis in Balb/c mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Urtica dentata Hand (UDH), the root of Laportea bulbifera (Sieb. et. Zucc.) Wedd, has long been utilized in traditional Chinese medicine for the treatment of rheumatoid arthritis and some other autoimmune diseases. Coumarins are the main active principles contributing to UDH's efficacy, but the mechanisms have not been fully clarified. AIM OF STUDY: To explore effects of total coumarins (TC) isolated from UDH on the development of type II collagen (CII)-induced arthritis (CIA) in Balb/c mice. MATERIALS AND METHODS: Arthritis was induced in Balb/c mice by immunization with an emulsion of 200mg CII and complete Freund's adjuvant (CFA). The CIA mice were then given with a suspension of TC or saline by intragastric (i.g.) administration every other day. The incidence and severity of disease and histopathology of inflammation were assessed. Inflammatory response was determined by measuring the levels of different inflammation mediators in serum. The effect of TC on differentiation of CD4(+)CD25(+) Foxp3(+)Treg cells was examined by flow cytometry. The phenotype of bone marrow-derived dendritic cells (DCs), T-bet mRNA level and IL-12p70 secretion by DCs were also detected. RESULTS: Pharmacologically, treatment with TC for type II collagen induced arthritis in mice through oral administration displayed significant and dose-dependent drop of clinical arthritis score and paw swelling, compared with the untreated CIA mice. Pathologic changes showed that TC protected tissues against bone destruction, whereas an almost complete destruction occurred in the CIA model group. The protective status was associated with a substantial decrease in the production of IFN-γ and IL-2, an increase of IL-10 and TGF-ß and suppressive expression of T-bet in DCs. TC also induced the generation of CD4(+)CD25(+) Treg cells with a Treg phenotype Foxp3. TC-treated DCs were characterized as low expression of MHC class II and CD86 molecules, as well as a reduction of IL-12p70. CONCLUSIONS: Our data suggest that TC provides substantial therapeutic protection against CIA by eliciting immune tolerance and it would be a valuable candidate for further investigation as a new anti-arthritic agent.