Metabolome and Genome Analysis of a Novel Endophytic Fungus Aureobasidium pullulans KB3: Discovery of Polyketones and Polyketone Biosynthesis Pathway.

Endophytic fungi associated with plants may contain undiscovered bioactive compounds. Under standard laboratory conditions, most undiscovered compounds are inactive, whereas their production could be stimulated under different cultivation conditions. In this study, six endophytic fungi were isolated from the bark of Koelreuteria paniculata in Quancheng Park, Jinan City, Shandong Province, one of which was identified as a new subspecies of Aureobasidium pullulans, named A. pullulans KB3. Additionally, metabolomic tools were used to screen suitable media for A. pullulans KB3 fermentation, and the results showed that peptone dextrose medium (PDM) was more beneficial to culture A. pullulans KB3 for isolation of novel compounds. Sphaerolone, a polyketone compound, was initially isolated from A. pullulans KB3 via scaled up fermentation utilizing PDM. Additionally, the whole-genome DNA of A. pullulans KB3 was sequenced to facilitate compound isolation and identify the biosynthesis gene clusters (BGCs). This study reports the multi-omics (metabolome and genome) analysis of A. pullulans KB3, laying the foundation for discovering novel compounds of silent BGCs and identifying their biosynthesis pathway.

lncRNA028466 regulates Th1/Th2 cytokine expression and associates with Echinococcus granulosus antigen P29 immunity.

BACKGROUND: Cystic echinococcosis (CE) is a parasitic disease that is caused by Echinococcus granulosus (Eg). The recombinant Echinococcus granulosus antigen P29 (rEg.P29) was shown to confer effective immunity to sheep and mice during E. granulosus secondary infection in our previous study. In this study, we sought to investigate the ability of long noncoding RNA 028466 (lncRNA028466) as a regulator for the protective immunity mediated by rEg.P29 vaccination and to study the effects of lncRNA028466 on CD4+T cell differentiation in mice spleen. METHODS: Female BALB/c mice were divided into two groups and were vaccinated subcutaneously with rEg.P29 antigen and PBS as a control (12 mice each group). Following prime-boost vaccination, CD4+T, CD8+T, and B cells from the spleen were isolated by flow cytometry. Quantitative real-time PCR (qRT-PCR) was performed to measure the expression of lncRNA028466 in these three kinds of cells. Then, lncRNA028466 was overexpressed and knocked down in naive CD4+T cells, and Th1 and Th2 cytokine expression was detected. qRT-PCR, western blot, and ELISA were performed to evaluate the production of IFN-γ, IL-2, IL-4, and IL-10, and flow cytometry was performed to detect the differentiation of Th1 and Th2 subgroups. RESULTS: lncRNA028466 was significantly decreased after the second week of immunization with rEg.P29 antigen. The proportion of CD4+ T cells was increased after rEg.P29 immunization. Overexpression of lncRNA028466 facilitated the production of IL-4, IL-10 and suppressed the production of IFN-γ, IL-2. Furthermore, after transfection with siRNA028466, IL-2 production was facilitated and IL-10 production was suppressed in naive CD4+ T cells. CONCLUSIONS: Immunization with rEg.P29 downregulated the expression of lncRNA028466, which was related to a higher Th1 immune response and a lower Th2 immune response. Our results suggest that lncRNA028466 may be involved in rEg.P29-mediated immune response by regulating cytokine expression of Th1 and Th2.

[Effect of Recombinant Antigen Ferritin on DC Induce Immune Response].

OBJECTIVE: To explore the immunoreaction against protoscolex infection by the recombinant ferritin of echinococcus granulosus (rEg.ferritin) immunized mice. METHODS: Bone marrow deriedstem cells (BMDCs) were collected and exposed in different antigens by cell culture and included 6 groups,there were hydatid fluid (HF) group (HF 30 µg/mL),rEg.ferritin group (rEg.ferritin 1 µg/mL), rEg.ferritin+LPS group (rEg.ferritin 1 µg/mL and LPS 100 ng/mL),HF+LPS group (HF 30 µg/mL and LPS 100 ng/mL),LPS group (LPS 100 ng/mL) and control (not add antigen). Morphology of BMDCs in different groups were detected by scan electron microscope (SEM). Phagocytic-ability,T cell multiplication capability and surface marker expression of BMDCs in different groups were detected by flow cytometry.Cytokines of Th1 [interleukin (IL)-12p70,tumor necrosis factor-α (TNF-α)] and Th2 (IL-6 and IL-10) in surpernatant of BMDCs in different groups were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: rEg.ferritin or rEg.ferritin+LPS stimulated BMDCs maturation, which the dendrites number of BMDCs in these 2 groups were more and longer than control ,HF and HF+LPS groups ( P<0.05), and induced higher levels of surface molecules major histocompatibility complexⅡ (MHCⅡ),CD80,CD86 and CD40,also had strong ability to induce T cell multiplication than control,HF,HF+LPS groups ( P<0.05),but had weak phagocytosis than HF group,and secreted higher levels of IL-6,IL-12p70,TNF-α and IL-10 than control group. CONCLUSION: BMDCs are stimulated with rEg.ferritin and became maturation. By activating T cell and releasing more cytokines of Th1 and Th2,rEg.ferrtin can induce BMDCs to produce Th1 and Th2 immunoreaction.

[Cloning, Expression and Immungenicity Analysis of Antigen Eg-01883 Screened from Protoscoleces of Echinococcus granulosus].

Objective: To screen for the Echinococcus granulosus 01883（Eg-01883） specifically expressed at the protoscolex period, clone and express this molecule as well as analyse its immunogenicity. Methods: Eg-01883, which is highly expressed at the protoscolex period but not in oncosphere， was screened by analysing the published mRNA sequences of E. granuolosus. Total RNA of E. granuolosus was extracted, Eg-01883 was cloned by RT-PCR, and the recombinant plasmid pET28a-Eg-01883 was constructed. Expression of the recombinant protein rEg-01883 was induced by isopropyl-ß-D-thiogalactoside （IPTG）. ICR mice were randomized into 3 groups （n=12 in each group）. Mice in the immunization group received subcutaneous injections of 10 µg rEg-01883 in 100 µl PBS emulsified in Freund's adjuvant at multiple sites, followed by immune enhancement after 2 weeks. Mice in the adjuvant group were injected with PBS and adjuvant. Mice in the control group received no treatment. Blood was obtained through caudal vein before immunization, and at 1, 2, and 4 weeks after the first immunization, and through the eyeball at 6 weeks after immunization. Serum levels of IgG, IFN-γ and IL-4 were determined by ELISA. The immunogenicity of rEg-01883 was identified by Western blotting. Results: Eg-01883 was screened, cloned, expressed and purified to obtain the recombinant protein rEg-01883, which mainly existed as the inclusion body. ELISA results showed that immunization with rEg-01883 induced production of specific IgG antibody. The serum IgG level in the immunization group increased from 1 week after the first immunization, peaked at 6 weeks（2.344±0.153）, which was significantly higher than those of the adjuvant group（0.206 1±0.006） and the control group （0.241±0.01） （P<0.01）. At 6 weeks after the first immunization, the serum levels of IFN-γ （43.23 pg/ml） and IL-4（24.88 pg/ml） in the immunization group were significantly higher than those in the adjuvant group（21.77 pg/ml, 13.27 pg/ml） and the control group（17.40 pg/ml, 12.25 pg/ml）（P<0.05）. Western blot showed that the recombinant protein rEg-01883 could be recognized by His-Tag antibodies, serum of immunized mice, and serum of mice with secondary infection. Conclusion: The recombinant protein rEg-01883 shows good immunogenicity in ICR mice.

Four heat shock protein genes of the endoparasitoid wasp, Cotesia vestalis, and their transcriptional profiles in relation to developmental stages and temperature.

Heat shock proteins (Hsps) play important roles in the environmental adaptation of various organisms. To explore the functions of Hsps in relation to heat stress and development in Cotesia vestalis, a solitary larval endoparasitoid of Plutella xylostella, four heat shock protein genes, CvHsp40, CvHsc70, CvHsp70 and CvHsp90, were cloned and sequenced from C. vestalis by real-time quantitative PCR and RACE. The cDNA sequence of CvHsp40, CvHsc70, CvHsp70 and CvHsp90 were 1473 bp, 2316 bp, 2279 bp and 2663 bp long, which encode proteins with calculated molecular weights (MW) of 39.1 kDa, 71.2 kDa, 70.1 kDa and 83.3 kDa, respectively. Furthermore, the analysis of genomic DNA confirmed that no introns existed in CvHsp40, CvHsp70 and CvHsp90 while two introns were present in CvHsc70. The amino acid sequence analysis of CvHsps indicated that CvHsp40 is a Type II Hsp40 homolog, CvHsp70 and CvHsc70 are the eukaryotic cytoplasmic Hsp70s, and CvHsp90 is the ß-isoform of Hsp90. The divergent transcriptional patterns of CvHsp40, CvHsp70 and CvHsp90 in the different developmental stages suggested that CvHsp transcripts were under different mechanisms of regulation during the development of parasitoid larvae. The dramatic increase of transcripts of CvHsp70 at the third-instar larva coincided with its developmental change in this stage, that is, from inside host to outside host. CvHsp40, CvHsc70 and CvHsp70 showed a trend of sex-specific differences of transcript abundance in the adult stage. All CvHsp transcripts in different developmental stages were significantly induced by heat stress, and the lowest transcript abundances appeared around the temperature 27°C, which probably suggest that this is the most favorable temperature for the development of C. vestalis. Our results suggest that the expression of heat shock proteins reflects to some extent the developmental changes and environmental requirements of insects.

[Relationship between occupational stress and type 2 diabetes mellitus].

OBJECTIVE: To investigate the association of occupational stress and related factors with type 2 diabetes mellitus (T2MD). METHODS: In case-control study, a questionnaire survey was conducted in 201 T2MD patients and 201 controls, who were selected from the staff members of organizations, enterprises, and institutions in Ningxia Hui Autonomous Region, China according to inclusion and exclusion criteria, to acquire the information on general condition and occupational stress. These subjects also underwent physical examinations and blood biochemical analyses. RESULTS: The T2MD group had significantly higher total occupational stress score, as well as the scores on such factors as workload, interpersonal relationship, and home/work balance than the control group (P < 0.01). After adjustment for age, gender, education level, smoking, and drinking, the odds ratios for T2MD were 2.538 and 3.075 in the people with moderate and severe stress, respectively, compared to those with mild stress. The risk factors for T2MD included drinking, family history of diabetes, waist circumference, triglyceride level, and total occupational stress score, while the protective factors included educational level and high-density lipoprotein level. CONCLUSION: Occupational stress is associated with the incidence of T2MD; the higher the degree of stress, the greater the risk of T2MD. Relevant measures should be taken to reduce the occupational stress or improve the ability of workers to cope with the stress, thus decreasing the incidence of T2MD among occupational population.

[Preliminary study on the difference in immune protection between Echinococcus granulosus recombinant ferritin and recombinant mMDH].

OBJECTIVE: To evaluate the immunoprotective activity of the Egrecombinant ferritin and Egrecombinant mMDH proteins in mice. METHODS: Thirty ICR mice were divided into 3 groups and immunized by injection of adjuvant-emulsified rEgferritin, rEgmMDH and PBS, respectively, in multiple spots at back, for 3 times with an interval of 2 wk. Two weeks after the last immunization, the mice of the 3 groups were infected intraperitoneally with 0.1 ml suspension containing about 1 500 Echinococcus granulosus (Eg) protoscoleces. The mice were sacrificed 22 wk after infection and the Eg cysts were collected and measured. Spleens were taken for detecting CD4+ T cells and CD8+ T cells and ratio calculated. RESULTS: Eg cysts were found in 30% (3/10) of the mice in the rEgferritin group with 5 cysts altogether; cysts were received in all the mice in the rEgmMDH group with 118 cysts totally; and cysts were found in 7 of 9 mice in the PBS control with 35 cysts totally. The mice in the rEgferritin group showed an 84.7% protection but revealed no protection in the rEgmMDH group. The CD4+ T cells were significantly higher in the rEgferritin group than the control, but no statistical difference was found in CD8+ T cells and CD4+/CD8+ ratio between the 2 groups. There was no considerable change in the T cells and ratio in the rEgmMDH group compared to the control. CONCLUSION: The Egrecombinant ferritin can inhibit the growth of Eg while the Egrecombinant mMDH seems promoting its growth in mice.

[Epidemiology of uterine corpus cancer in some cancer registering areas of China from 2003 - 2007].

OBJECTIVE: To analyze corpus uteri epidemiology in selected cancer registering areas of China during 2003 - 2007, and to provide scientific information for its prevention and control in China. METHODS: The incident and mortality data of corpus uteri cancer in 32 cancer registering areas of China with better quality during 2003 - 2007, which were selected according to the criteria of and provided by National Center for Cancer Registration, were analyzed. RESULTS: There were 8850 new cases and 1559 death cases of corpus uteri cancer, which accounted for 2.96% (8 850/299 306) of all female new cancer cases and 0.94% (1 559/166 305) of all female cancer death cases, respectively. Corpus uteri cancer was the 9th most common cancer for all new female cancer cases which world age adjusted incidence rates was 5.04/105, and 19th most common cancer for all female cancer death cases which world age adjusted mortality rate was 0.83/105 in 32 selected cancer registering areas of China during 2003 - 2007. Zhongshan city, Guangzhou city in Guangdong province and Beijing were the areas with the highest incidence rates in which were 14.51/105, 8.51/105 and 6.69/105, respectively. Zhongshan city in Guangdong province, Dafeng city in Jiangsu province and Feicheng city in Shandong province were the areas with the highest mortality rates, in which were 4.03/105, 3.19/105 and 1.65/105 respectively during 2003 - 2007. There were increasing trends for its incidence rates in above 32 areas during 2003 - 2007, its world age adjusted incidence rates increased from 3.94/105 in 2003 to 5.56/105 in 2007 (P = 0.026), while its urban world age adjusted incidence rates increased from 4.57/105 in 2003 to 6.18/105 in 2007 (P = 0.038), and rural rates increased from 1.74/105 in 2003 to 3.01/105 in 2007 (P = 0.013), and the results showed that urban areas obviously higher than rural areas (P < 0.01). Although there was a slow increasing trend for its world age adjusted mortality rates in above 32 areas during 2003 - 2007 which increased from 0.64/105 in 2003 to 0.87/105 in 2007 (P = 0.214), and from 0.66/105 in 2003 to 0.88/105 in 2007 in urban areas (P = 0.340), and from 0.57/105 in 2003 to 0.83/105 in 2007 in rural areas (P = 0.070), while increasing trends without statistical significance.But mortality rates in urban areas were obviously higher than those of rural areas (P < 0.01). CONCLUSIONS: Although the world standardized incidence and mortality rates of corpus uteri cancer were at low level worldwide, there were increasing trends for its incidence rates during 2003 - 2007 in the 32 selected cancer registering areas of China. Moreover, its incidence and mortality rates were at high level worldwide in some areas such as Zhongshan city of Guangdong province and Dafeng city of Jiangsu province during the period, in which suggested that its prevention and control should be enhanced.

Echinococcus granulosus 14-3-3 protein: a potential vaccine candidate against challenge with Echinococcus granulosus in mice.

OBJECTIVE: To investigate the protective immunity against Echinococcus granulosus in mice immunized with rEg14-3-3. METHODS: ICR mice were subcutaneously immunized three times with rEg14-3-3, followed by the challenge with Echinococcus granulosus protoscoleces intraperitoneally and then sacrificed after six months of post-challenge to detect the proliferation of splenocytes by MTT assay, and to measure the secretion of IL-2, IL-4, IL-10, and IFN-γ by ELISA. The rate of reduced hydatid cyst and the levels of IgE, IgG and IgG subclasses in sera were examined. RESULTS: Mice vaccinated with rEg14-3-3 and challenged with protoscoleces revealed significant protective immunity of 84.47%. ELISA analysis indicated that the immunized mice generated specific high levels of IgG and the prevailing isotypes of IgG were IgG1 and IgG2a. Splenocytes from mice immunized with rEg14-3-3 showed a significant proliferation response. The secretion of IFN-γ and IL-2 increased significantly in the vaccinated mice whereas there was no significant difference in IL-4 and IL-10 levels between vaccinated and control mice. CONCLUSION: The results indicate that the rEg14-3-3 vaccine could induce a high level of protective immunity as a promising vaccine candidate to prevent cystic echinococcosis.

[Study on immune response in ICR mice by immunization with recombinant myophilin vaccine against Echinococcus granulosus].

OBJECTIVE: To investigate the immune response and the protection in mice induced by the recombinant myophilin protein of Echinococcus granulosus. METHODS: Thirty-six male ICR mice of 6-8 weeks old were randomly divided into groups A, B and C each with 12. The mice in the 3 groups were subcutaneously immunized with Eg myophilin protein, blank plasmid protein or PBS, respectively, by 3 times and challenged with protoscoleces of E. granulosus 2wk after the last vaccination. Mice were sacrificed 20wk after the infection, the hydatid cysts were collected for measuring the weight reduction. Spleens were obtained and the splenocytes were separated and cultured in vitro with EgAg or ConA stimulus for 4-5 h. The subsets of CD4+ and CD8+ T cells were measured by FACsort. The proliferation of splenocytes was determined by MTT method with blank plasmid and PBS as control. RESULTS: The average weight of the hydatid cysts in the immunized group decreased by 69.1% in comparison to the blank plasmid and PBS groups. The CD4+ subset [(29.7 +/- 0.9)%] and CD84+ subset [(9.7 +/- 0.8)%] in group A increased significantly than group C, [(11.6 +/- 1.4)%] and [(7.8 +/- 0.2)%] respectively (P < 0.01 or < 0.05). The ratio of CD4+/CD8+ subsets in group A (3.061 +/- 0.015) was also higher than group C (1.487 +/- 0.106) (P < 0.01). Without stimulation, the proliferation of T lymphocytes in group A(0.237 +/- 0.009) was higher than group C (0.159 +/- 0.005) (P < 0.01), with EgAg or ConA stimulus, it was also higher in group A than that of group C (P < 0.01). CONCLUSION: The recombinant myophilin protein of E. granulosus can induce the proliferation of splenocytes and Th1 response in mice, and the CD4+ T cells subset may bear a part in the induced protection against the challenge of protoscoleces.

Stereoselective synthesis of desloratadine derivatives as antagonist of histamine.

A series of desloratadine derivatives were stereoselectively synthesized and evaluated for H(1) antihistamine activity. For the evaluation of H(1) antihistamine activity, the in vitro histamine-induced contraction of the guinea-pig ileum assay (HC) was used. The synthesized desloratadine derivatives 7, 8 and 9 are structurally related to rupatadine and were generated by replacement of the 5-methyl-3-pyridine group of rupatadine with gamma-alkylidene butenolide. Their H(1) antihistamine activities have shown a high dependence on the exact nature of the substituent in the lactone ring. Optimum structures 7, 8a and 8g display potent activity inhibiting histamine-induced effects.

[Cloning, expression and immunologic identification of Eg10 gene of Echinococcus granulosus].

OBJECTIVE: To clone and express Eg10 gene of Echinococcus granulosus, and investigate the immunological characteristic of the recombinant. METHODS: Eg10 gene was subcloned into pET28a vector. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. The recombinant protein was purified with His-bind purification kit. Forty-eight mice were randomly divided into 4 groups. Mice in groups A and B were injected with PBS and PBS+Freund's adjuvant (100 microl) as control. Mice in groups C and D were immunized with 10 mg and 50 microg purified Eg10 antigen formulated in Freund's adjuvant, respectively. All the mice received three immunizations at 2-week intervals with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization. The immunological characteristics of recombinant Eg10 was analyzed by Western blotting and ELISA. RESULTS: The recombinant Eg10 protein (Mr 31 000) was expressed in E. coli BL21. The recombinant Eg10 and expression product of PET28a/Eg10 were recognized by sera from mice immunized with recombinant Eg10. ELISA showed that the titer of IgG reached a peak at the 8th week in groups C and D, the level of IgG in sera of groups C or D was higher than that of groups A or B (P < 0.05) at the 2nd, 4th, 6th, 8th, and 10th week. There was no significant difference in the level of IgG between group C and group D (P > 0.05). CONCLUSION: The Eg10 gene has been expressed with immunogenicity.