Trichoderma panacis sp. nov., an endophyte isolated from Panax notoginseng.

An endophytic member of the genus Trichoderma was isolated from the root of a healthy 3-year-old Panax notoginseng in Yunnan province, PR China. The results of phylogenetic analyses based on a combined of ITS, tef1 and rpb2 indicated that this isolate was distinct from other species of the genus Trichoderma and closely related to Trichoderma songyi. It can be distinguished from T. songyi by its slower growth rates on PDA and colony morphology. The novel isolate formed conidia in thick white pustules scattered mostly at the margin. Its conidiophores tended to be regularly verticillium-like, little branched, sometimes substituted by phialides singly or in whorls. Conidia are smooth, mostly broadly subglobose to ellipsoidal. In combination with the genotypic and phenotypic characteristics, all data demonstrated that the fungus studied represented a unique and distinguishable novel species of the genus Trichoderma, for which the name Trichoderma panacis sp. nov. is proposed.

[Synthesis of triterpenoid saponins from Aesculus chinensis based on transcriptome data].

Aesculus chinensis belongs to Hippocastanaceae family,bears medicinal and ornamental values. The oleanane type triterpenoid saponin aescin is regarded as active ingredient and accumulated in seed. In order to understand its molecular basis of the triterpenoid biosynthesis,we used high-throughput sequencing under Illumina Hi Seq 2000 platform to obtain the transcriptome data of seed and flower from A. chinensis to further mine the genes involved in its metabolic pathway. Unigene's de novo splicing was performed using Trinity software; the transcriptome results were annotated with KEGG database to predict the specific pathways of the aescin triterpenoid metabolism. Terpenoid and triterpenoid pathways were found from transcriptome data,and forty seven and twenty seven corresponding genes were uncovered respectively. It was found that there are eight kinds of enzymes related to the terpenoid metabolism pathway precursors and three kinds of enzymes related to the triterpenoid metabolism pathway. In this study,five genes corresponding to triterpene cyclase were analyzed in A. chinensis for the first time,which may participate in the synthesis of triterpenoid. It' s revealed that there were thirty three differential genes associated with the ko00900 and ko00909 pathways by analysis on the difference in transcriptome expression between seeds and flowers; seventeen unigenes were up-regulated and sixteen unigenes were down-regulated in the seeds relative to flowers. In this study, qRT-PCR experiments were used to verify the expression of three key enzyme genes of SQE( Unigene25806),HMGS( Unigene36710),and ß-AS( Unigene33291). The results of qRT-PCR were consistent with the transcriptome data. The candidate genes related to triterpenoid saponin aescin synthesis in A. chinensis found in this study can provide theoretical basis for the metabolism synthesis and regulation of aescin.

Drechmeria panacis sp. nov., an endophyte isolated from Panax notoginseng.

An endophytic strain (designated as strain SYPF 8335T) was isolated from a root of Panax notoginseng in Wenshan district, Yunnan province of China. Strain SYPF 8335T grew very slowly and formed white colonies. Phylogenetic analysis of four loci indicated that strain SYPF 8335T was placed in the Drechmeria clade with Drechmeria campanulata as its closest phylogenetic neighbour. The nucleotide differences between strain SYPF 8335T and D. campanulata are 30 substitutions in the internal transcriber region region. A key morphological feature that differentiates the two fungi is that D. campanulata produces campanulate conidia. Combined with the morphology and molecular analyses, a new species named Drechmeria panacis sp. nov., is proposed.

Verruconis panacis sp. nov., an endophyte isolated from Panax notoginseng.

An endophytic strain (designated as strain SYPF 8337T) was isolated from the root of 3-year-old Panax notoginseng in Yunnan province of China. Strain SYPF 8337T grew slowly and formed pale brown to brown colonies. Phylogenetic analyses indicated that strain SYPF 8337T was placed in the Verruconis clade. Different from other Verruconis species, strain SYPF 8337T produced four-cell conidia. Furthermore, strain SYPF 8337T is the first fungus isolated as an endophyte of P. notoginseng in the genus Verruconis. Combined with the morphology and molecular analyses, a new species named Verruconis panacis sp. nov. is proposed.

Absidia panacisoli sp. nov., isolated from rhizosphere of Panax notoginseng.

A strain (SYPF 7183T) was isolated from rhizosphere soil of Panax notoginseng in southwest China. Phylogenetic analyses indicated that strain SYPF 7183T was distinct from the other Absidia species with well-supported values. Strain SYPF 7183T produced spherical or subpyriform sporangia and short cylindrical sporangiospores. The azygospores were globose to oval. Based on morphological and phylogenetic evidence, the novel strain Absidia panacisoli sp. nov. is proposed.

Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters.

BACKGROUND: 2-Keto-L-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, L-sorbose is transformed into 2-KGA by the symbiosis system composed of Ketogulonicigenium vulgare and Bacillus megaterium. Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization. RESULTS: In this study, Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. The complete genome of K. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported K. vulgare genomes, K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (orf_01408 promoter) and P2 (orf_02221 promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into K. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimization, the recombinant strain K. robustum/pBBR-P1_xfp2502-P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strain K. robustum/pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd) and pyruvate dehydrogenase genes (pdh) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh) and sorbosone dehydrogenase gene (sndh), were up-regulated to different degrees in the recombinant strain. CONCLUSIONS: The genome-based functional analysis of K. robustum SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production.

Xylarianins A-D from the endophytic fungus Xylaria sp. SYPF 8246 as natural inhibitors of human carboxylesterase 2.

Eighteen secondary metabolites were isolated from the fermentation broth of the endophytic fungus Xylaria sp. SYPF 8246, including four new compounds, xylarianins A-D (1-4), three new natural products, 6-methoxycarbonyl-2'-methyl-3,5,4',6'-tetramethoxy-diphenyl ether (5), 2-chlor-6-methoxycarbonyl-2'-rnethyl-3,5,4',6'-tetramethoxy-diphenyl ether (6), and 2-chlor-4'-hydroxy-6-methoxy carbonyl-2'-methyl-3,5,6'-trimethoxy-diphenyl ether (7), and eleven known compounds (8-18). Their structural elucidations were conducted by using 1D and 2D NMR, HRESIMS, and Rh2(OCOCF3)4-induced electronic circular dichroism (ECD) spectra analyses. The integrated 1H and 13C NMR data of three new natural products 5-7 were reported for the first time. All the isolated compounds were assayed for their inhibitory activities against human carboxylesterase 2 (hCE 2). Compounds 1, 5-9, and 18 displayed significant inhibitory activities against hCE 2 with IC50 values of 10.43 ±â€¯0.51, 6.69 ±â€¯0.85, 12.36 ±â€¯1.27, 18.25 ±â€¯1.78, 29.78 ±â€¯0.48, 18.86 ±â€¯1.87, and 20.72 ±â€¯1.51 µM, respectively. The interactions between compounds 1 and 5 with hCE 2 were anaylzed by molecular docking.

Drechmerin H, a novel 1(2), 2(18)-diseco indole diterpenoid from the fungus Drechmeria sp. as a natural agonist of human pregnane X receptor.

A novel 1(2), 2(18)-diseco indole diterpenoid, drechmerin H (1), was isolated from the fermentation broth of Drechmeria sp. together with a new indole diterpenoid, 2'-epi terpendole A (3), and a known analogue, terpendole A (2). Their structures were determined by HRESIMS, 1D and 2D NMR, ECD, and X-ray single crystal diffraction analyses as well as quantum chemical calculation. The abosulte configuration of terpendole A (2) was determined for the first time. Compound 1 displayed the significant agonistic effect on pregnane X receptor (PXR) with EC50 value of 134.91 ±â€¯2.01 nM, and its interaction with PXR was investigated by molecular docking. Meantime, a plausible biosynthetic pathway for compounds 1-3 is also discussed in the present work.

Serosurvey of Avian metapneumovirus, Orithobacterium rhinotracheale, and Chlamydia psittaci and Their Potential Association with Avian Airsacculitis.

Seasonal outbreaks of airsacculitis in China's poultry cause great economic losses annually. This study tried to unveil the potential role of Avian metapneumovirus (AMPV), Ornithobacterium rhinotracheale (ORT) and Chlamydia psittaci (CPS) in avian airsacculitis. A serological investigation of 673 breeder chickens and a case-controlled study of 430 birds were undertaken. Results showed that infection with AMPV, ORT, and CPS was highly associated with the disease. The correlation between AMPV and CPS were positively robust in both layers and broilers. Finally, we determined the co-infection with AMPV, ORT, and CPS was prevalent in the sampled poultry farms suffering from respiratory diseases and the outbreak of airsacculitis was closely related to simultaneous exposure to all three agents.

Ir-Catalyzed Asymmetric Total Synthesis of (-)-Communesin F.

The first catalytic asymmetric total synthesis of the heptacyclic alkaloid (-)-communesin F is described. A key step features an iridium-catalyzed asymmetric intermolecular cascade cyclization, constructing the lower N,N-aminal-containing CDEF tetracyclic core in one step. Another notable element is the closure of final ring system (A ring) via a facile reduction of a twisted amide and concomitant cyclization activated by mesylation of N,O-hemiaminal intermediate.

Bacillus notoginsengisoli sp. nov., a novel bacterium isolated from the rhizosphere of Panax notoginseng.

A Gram-stain-positive, rod-shaped, motile bacterium designated as SYP-B691T was isolated from rhizospheric soil of Panax notoginseng. Phylogenetic analysis indicated that SYP-B691T clearly represented a member of the genus Bacillus and showed 16S rRNA gene similarity lower than 97.0 % with the type strains of species of the genus Bacillus, which indicates that it should be considered as a candidate novel species within this genus. The optimum growth of the strain was found to occur at 37 °C and pH 7.0-9.0. The genomic DNA G+C content was determined to be 45.2 mol%. It contained meso-2,6-diaminopimelic acid in the cell-wall peptidoglycan. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unknown phospholipid. MK-7 was the only menaquinone identified. The major cellular fatty acids of SYP-B691T were identified as iso-C15 : 0 and anteiso-C15 : 0. On the basis of phenotypic, chemotaxonomic and phylogenetic characteristics, SYP-B691T merits recognition as a representative of a novel species of the genus Bacillus, for which the name Bacillus notoginsengisoli sp. nov. is proposed, with SYP-B691T(=DSM 29196T=JCM 30743T) as the type strain.

Arthrobacter ginkgonis sp. nov., an actinomycete isolated from rhizosphere of Ginkgo biloba L.

A Gram-stain-positive, aerobic actinobacterial strain (designated SYP-A7299T), which displayed a rod-coccus growth lifecycle, was isolated from the rhizosphere of Ginkgo biloba L. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain SYP-A7299T belongs to the genus Arthrobacter and is most closely related to Arthrobacter halodurans JSM 078085T (97.4 % 16S rRNA gene sequence similarity). The DNA-DNA relatedness value between strain SYP-A7299T and A. halodurans JSM 078085T was 37 % ±2.9. The cell-wall peptidoglycan was A4α, and glucose and galactose were whole-cell sugars. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, two glycolipids and an unknown polar lipid. The major menaquinone were MK-8(H2) (72 %) and MK-9(H2) (28 %), and the predominant cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content was 68.9 mol%. Based on the morphological, physiological, biochemical and chemotaxonomic characters presented in this study, strain SYP-A7299T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter ginkgonis sp. nov. is proposed. The type strain is SYP-A7299T (=DSM 100491T=KCTC 39 592T).

Microalgae-based advanced municipal wastewater treatment for reuse in water bodies.

Reuse of secondary municipal effluent from wastewater treatment plants in water bodies could effectively alleviate freshwater resource shortage. However, excessive nutrients must be efficiently removed to prevent eutrophication. Compared with other means of advanced wastewater treatment, microalgae-based processes display overwhelming advantages including efficient and simultaneous N and P removal, no requirement of additional chemicals, O2 generation, CO2 mitigation, and potential value-added products from harvested biomass. One particular challenge of microalgae-based advanced municipal wastewater treatment compared to treatment of other types of wastewater is that concentrations of nutrients and N:P ratios in secondary municipal effluent are much lower and imbalanced. Therefore, there should be comprehensive considerations on nutrient removal from this specific type of effluent. Removal of nutrients and organic substances, and other environmental benefits of microalgae-based advanced municipal wastewater treatment systems were summarized. Among the existing studies on microalgal advanced nutrient removal, much information on major parameters is absent, rendering performances between studies not really comparable. Mechanisms of microalgae-based nitrogen and phosphorus removal were respectively analyzed to better understand advanced nutrient removal from municipal secondary effluent. Factors influencing microalgae-based nutrient removal were divided into intrinsic, environmental, and operational categories; several factors were identified in each category, and their influences on microalgal nutrient removal were discussed. A multiplicative kinetic model was integrated to estimate microalgal growth-related nutrient removal based majorly on environmental and intrinsic factors. Limitations and prospects of future full-scale microalgae-based advanced municipal wastewater treatment were also suggested. The manuscript could offer much valuable information for future studies on microalgae-based advanced wastewater treatment and water reuse.

Luteimonas notoginsengisoli sp. nov., isolated from rhizosphere.

A Gram-staining-negative, yellow-pigmented strain, designated SYP-B804T, was isolated from the rhizosphere of Panax notoginseng. The strain was rod-shaped with a single polar flagellum. The optimum temperature and pH required for growth of the strain were 28-32 °C and pH 7-8, respectively. 16S rRNA gene sequence analysis indicated that strain SYP-B804T showed highest 16S rRNA gene sequence similarity with Luteimonas mephitis DSM 12574T (98.0 %). However, the DNA-DNA relatedness value between them (38.1 ± 0.6 %) was less than the threshold value for the delineation of genomic species. Ubiquinone-8 (Q-8) was the predominant quinone. The major fatty acids were iso-C15 : 0 and iso-C17 : 1ω9c. The major polar lipids of the strain were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genomic DNA was 71 %. On the basis of phenotypic, chemotaxonomic and molecular characteristics, strain SYP-B804T merits recognition as a representative of a novel species of the genus Luteimonas, for which the name Luteimonas notoginsengisoli sp. nov. is proposed, with SYP-B804T ( = KCTC 42211T = JCM 30329T) as the type strain.

Nocardioides ginkgobilobae sp. nov., an endophytic actinobacterium isolated from the root of the living fossil Ginkgo biloba L.

A Gram-stain-positive, aerobic and yellow actinobacterial strain, designated SYP-A7303T, was isolated from the root of Ginkgo biloba L. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain SYP-A7303T belongs to the genus Nocardioides. The 16S rRNA gene sequence of strain SYP-A7303T showed highest similarity to Nocardioides marinus CL-DD14T ( = JCM 15615T) (98.3 %) and Nocardioides aquiterrae GW-9T ( = JCM 11813T) (97.1 %), and less than 96.9 % to the type strains of other species of the genus Nocardioides. Strain SYP-A7303T grew optimally at 28 °C, pH 7.0 and in the absence of NaCl. It contained ll-2,6-diaminopimelic acid in the cell-wall peptidoglycan, with mannose, ribose, rhamnose, glucose and galactose as whole-cell sugars. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and an unknown lipid. The menaquinone was MK-8(H4) and the predominant cellular fatty acids were iso-C16 : 0, C18 : 1ω9c and C17 : 1ω8c. The DNA G+C content was 72 mol%. Mean DNA-DNA relatedness values between strain SYP-A7303T and the closely related strains N. marinus JCM 15615T and N. aquiterrae JCM 11813T were 62.5 ± 2.4 and 56.5 ± 3.5 %, respectively. Based on the morphological, physiological, biochemical and chemotaxonomic characteristics presented in this study, strain SYP-A7303T represents a novel species of the genus Nocardioides, for which the name Nocardioides ginkgobilobae sp. nov. is proposed. The type strain is SYP-A7303T ( = DSM 100492T = KCTC 39594T).

Development of a Novel PmpD-N ELISA for Chlamydia psittaci Infection.

OBJECTIVE: Chlamydia psittaci is an avian respiratory pathogen and zoonotic agent. The wide prevalence of C. psittaci poses a threat to the poultry industry and its employees. However, few commercial kits are available for detecting avian antibodies excluding the in-house ELISA kit. In this study, we developed a novel ELISA kit for detecting antibodies against C. psittaci based on the N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) as the coating antigen. METHODS: The antigen concentrations, primary antibody, and cut-off value were determined and optimized. The ELISA, designated PmpD-N ELISA, was assessed for sensitivity, specificity, and concordance using sera samples from 48 experimentally infected and 168 uninfected SPF chickens. RESULTS: The sensitivity and specificity of PmpD-N ELISA were 97.9%, 100%, respectively, while the concordance was 98.1% as compared to that of MOMP-ELISA. No cross-reaction with positive sera for other avian pathogens was found. Using PmpD-N ELISA, 799/836 clinical samples were positive, including 93.0% and 98.1% positivity in layers and broilers, respectively. CONCLUSION: These data indicate that indirect ELISA with PmpD-N as the antigen candidate is a promising approach for the surveillance of C. psittaci infection.

Flavobacterium notoginsengisoli sp. nov., isolated from the rhizosphere of Panax notoginseng.

Two novel bacterial strains, designated SYP-B540(T) and SYP-B556, were isolated from rhizospheric soil of Panax notoginseng located at Yunnan Province, China. Both strains were Gram-staining negative, aerobic, non-motile, elongated rod shaped and yellow coloured. They grew optimally at 28 °C and pH 7.0. Analysis of 16S rRNA gene sequences showed that the two strains shared 99.8 % sequence similarity to each other, but lower than 97.6 % to the other known species of the genus Flavobacterium. The predominant respiratory quinone for the two strains was MK-6, and the major fatty acids were iso-C15:0 and summed Feature 3 (comprising 16:1 ω7c and/or 16:1 ω6c). The polar lipids consisted of phosphatidylethanolamine, two unidentified polar lipids and three unidentified amino-phospholipids. The DNA G+C contents of strains SYP-B540(T) and SYP-B556 were 33.3 and 32.7 mol%, respectively. In addition, the DNA-DNA hybridization values of strains SYP-B540(T) and SYP-B556 to their closest phylogenetic neighbors were significantly lower than 70 %. On the basis of the polyphasic taxonomy studies, strains SYP-B540(T) and SYP-B556 represent a novel species of the genus Flavobacterium, for which the name Flavobacterium notoginsengisoli sp. nov. is proposed. The type strain is SYP-B540(T) (=KCTC 32505(T) = NBRC 110012(T) = BCRC 80724(T)).

Hepatic-targeted gene delivery using cationic mannan vehicle.

The incidence of hepatic diseases continuously increases worldwide and causes significant mortality because of inefficient pharmacotherapy. Gene therapy is a new strategy in the treatment of hepatic diseases, but the disadvantages of insufficient retention in the liver and undesirable side effects hinder its application. In this study, we developed a novel nonviral vehicle targeted to liver. Mannan was cationized with spermine at varying grafted ratios to deliver the gene and was optimized in cytotoxicity and transfection in vitro. A spermine-mannan (SM)-based delivery system was proven to be hepatic targeted and was capable of prolonging the gene retention period in the liver. Moreover, SM at N/P of 20 was confirmed to be less interfered with by the serum. Optimized SM vehicle has relatively high hepatic transfection with almost no toxicity induction in the liver, which highlighted its potential in the treatment of hepatic diseases.

Sinomonas notoginsengisoli sp. nov., isolated from the rhizosphere of Panax notoginseng.

A Gram-positive, aerobic, non-motile and pale yellow colour actinobacterial strain, designated SYP-B575(T), was isolated from rhizosphere soil of Panax notoginseng. The optimal growth of the strain was found to occur at 28 °C, pH 7.0 and without NaCl. Phylogenetic analysis indicated that strain SYP-B575(T) clearly belongs to the genus Sinomonas and should be considered as a candidate of novel species within this genus. The 16S rRNA gene sequence similarities between strain SYP-B575(T) and the other Sinomonas type strains ranged from 97.3 to 96.0%. The predominant isoprenoid quinone was identified as MK-9(H2) and the major fatty acids were identified as anteiso-C(15:0) and anteiso-C(17:0). The polar lipids were found to consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and glycolipids. The major cell-wall amino acids were identified as Lys, Ala, Glu, Gly and Ser. The whole-cell sugars were identified as mannose, ribose, rhamnose, glucose and galactose. The G+C content of the genomic DNA was determined to be 66.6 mol%. The DNA-DNA relatedness values between SYP-B575(T) and its closest phylogenetic neighbours were lower than 35.5%. On the basis of this polyphasic taxonomic study, strain SYP-B575(T) represents a novel species of the genus Sinomonas, for which the name Sinomonas notoginsengisoli sp. nov. is proposed. The type strain is SYP-B575(T) (=DSM 27685(T) = KCTC 29237(T)).

Gene recombinant bone marrow mesenchymal stem cells as a tumor-targeted suicide gene delivery vehicle in pulmonary metastasis therapy using non-viral transfection.

One of the main limitations of anti-tumor gene therapy is the lack of an effective way to deliver therapeutic genes to tumor sites. Bone marrow mesenchymal stem cells (BMSCs) have been proposed as cellular delivery vehicles to tumor sites in tumor-targeted cancer gene therapy. Here, we investigated the therapeutic effects of cytomegalovirus-thymidine kinase expressing BMSCs (TK-BMSCs) on pulmonary melanoma metastasis combined with prodrug ganciclovir. BMSCs were successfully engineered through a non-viral gene vector. The gene recombinant BMSCs migrated to the pulmonary area and were found to have the tendency to target tumor nodules after systemic delivery. In vitro results demonstrate that the engineered BMSCs have significant suicide effects in the presence of ganciclovir in a dose-dependent manner and can exert a sufficient bystander effect on B16F10 tumor cells in co-culture experiments. In vivo studies confirmed the therapeutic effects of TK-BMSCs/ganciclovir on the metastasis tumor model. FROM THE CLINICAL EDITOR: This study investigates the possibility of gene transfer via bone marrow mesenchymal stem cells in anti-cancer gene therapy using a metastatic melanoma model and cytomegalovirus-thymidine kinase expressing stem cells, demonstrating clear therapeutic effects.