[Effects of miR-203a targeting and its target gene on lymph node metastasis of breast cancer].

Objective: To study the expression and correlation of mir-203a and its target gene ATM in breast cancer tissues, so as to provide theoretical basis for the pathogenesis of breast cancer, especially lymph node metastasis. Methods: Thirty paired breast cancer and paracancer normal tissues were collected, and RT-qPCR was used to detect the relative expression levels of mir-203a and ATM in the samples of the two groups. Correlation analysis was conducted for mir-203a and ATM, and correlation analysis was conducted for the pathological characteristics, so as to compare whether there were statistical differences between mir-203a and ATM in lymph node metastasis and non-metastasis. Results: Compared with normal paracancer tissues, the expression level of mir-203a in breast cancer tissues was significantly increased (P＜0.01), and the expression level of ATM was significantly decreased (P＜0.01), showing a significant negative correlation between the two tissues (r=-0.847,P＜0.01).The expression level of mir-203a and ATM was significantly correlated with lymph node metastasis and different clinical stages (P＜0.05). The expression level of mir-203a in the group with lymph node metastasis were significantly lower than that in the group without lymph node metastasis (P＜0.05), and the expression of ATM in the group with lymph node metastasis was significantly higher than that in the group without lymph node metastasis (P＜0.01). Conclusion: The overexpression of mir-203a in the early stage of breast cancer may inhibit the expression of its target gene ATM, which may be a protective mechanism to regulate the proliferation,metastasis and invasiveness of tumor cells. In the middle and late stage, mir-203a is down-regulated and the ATM gene is up-regulated, which may be involved in lymph node metastasis.

Optimization of Fermentation Conditions and Bench-Scale for Improvement of a Novel Glycoprotein GP-1 Production by Streptomyces kanasenisi ZX01.

GP-1 is a novel glycoprotein produced by Streptomyces kanasenisi ZX01 that was isolated from soil near Kanas Lake with significant bioactivity against tobacco mosaic virus. However, extremely low fermentation production has largely hindered further research and market applications on glycoprotein GP-1. In this study, response surface methodology was used to optimize fermentation conditions in a shake flask for higher glycoprotein GP-1 production. When the optimized fermentation conditions were inoculum volume of 6%, initial pH of 6.5, and rotating speed of 150 rpm, glycoprotein GP-1 production could reach 0.9253 mg/L, which was increased by 52.14% compared to the original conditions. In addition, scale-up fermentation was conducted in a 5-L bioreactor to preliminarily explore the feasibility for mass production of glycoprotein GP-1 in a large fermentor, obtaining GP-1 production of 2.54 mg/L under the same conditions, which was 2.75 times higher than the production obtained from a shake flask of 0.9253 mg/L. This work will be helpful to improve GP-1 production on a large scale and lay the foundations for developing it as a novel agent against plant virus.

Effects of Agitation, Aeration and Temperature on Production of a Novel Glycoprotein GP-1 by Streptomyces kanasenisi ZX01 and Scale-Up Based on Volumetric Oxygen Transfer Coefficient.

The effects of temperature, agitation and aeration on glycoprotein GP-1 production by Streptomyces kanasenisi ZX01 in bench-scale fermentors were systematically investigated. The maximum final GP-1 production was achieved at an agitation speed of 200 rpm, aeration rate of 2.0 vvm and temperature of 30 °C. By using a dynamic gassing out method, the effects of agitation and aeration on volumetric oxygen transfer coefficient (kLa) were also studied. The values of volumetric oxygen transfer coefficient in the logarithmic phase increased with increase of agitation speed (from 14.53 to 32.82 h-1) and aeration rate (from 13.21 to 22.43 h-1). In addition, a successful scale-up from bench-scale to pilot-scale was performed based on volumetric oxygen transfer coefficient, resulting in final GP-1 production of 3.92, 4.03, 3.82 and 4.20 mg/L in 5 L, 15 L, 70 L and 500 L fermentors, respectively. These results indicated that constant volumetric oxygen transfer coefficient was appropriate for the scale-up of batch fermentation of glycoprotein GP-1 by Streptomyces kanasenisi ZX01, and this scale-up strategy successfully achieved 100-fold scale-up from bench-scale to pilot-scale fermentor.

Antifungal Activity and Action Mode of Cuminic Acid from the Seeds of Cuminum cyminum L. against Fusarium oxysporum f. sp. Niveum (FON) Causing Fusarium Wilt on Watermelon.

In order to develop a novel biofungicide, the antifungal activity and action mode of cuminic acid from the seed of Cuminum cyminum L. against Fusarium oxysporum f. sp. niveum (FON) on watermelon was determined systematically. In this study, the median effective concentration (EC50) value for cuminic acid in inhibiting mycelial growth of FON was 22.53 µg/mL. After treatment with cuminic acid, the mycelial morphology was seriously influenced; cell membrane permeability and glycerol content were increased markedly, but pigment and mycotoxin (mainly fusaric acid) were significantly decreased. Synthesis genes of bikaverin (Bike1, Bike2 and Bike3) and fusaric acid (FUB1, FUB2, FUB3 and FUB4) both were downregulated compared with the control, as confirmed by quantitative RT-PCR. In greenhouse experiments, cuminic acid at all concentrations displayed significant bioactivities against FON. Importantly, significant enhancement of activities of SOD, POD, CAT and decrease of MDA content were observed after in vivo cuminic acid treatment on watermelon leaves. These indicated that cuminic acid not only showed high antifungal activity, but also could enhance the self-defense system of the host plant. Above all, cuminic acid showed the potential as a biofungicide to control FON.

Two new Anti-TMV active chalconoid analogues from the root of Phyllanthus emblica.

Two new chalconoid analogues, emblirol A (1) and B (2), along with three known ones (3-5), were isolated from the root of the Phyllanthus emblica L. Their structures were elucidated on the basis of spectral data. Compound 1 and 2 showed moderate anti-TMV activity with inhibition rates 79.6 and 62.1% at a concentration of 1 mg/mL, respectively.

Optimization of medium compositions to improve a novel glycoprotein production by Streptomyces kanasenisi ZX01.

Streptomyces kanasenisi ZX01 was found to produce a novel glycoprotein GP-1 previously, which was secreted into medium and had significant activity against tobacco mosaic virus. However, the low production of GP-1 by strain ZX01 limited its further studies. In order to improve the yield of GP-1, a series of statistical experimental design methods were applied to optimize medium of strain ZX01 in this work. Millet medium was chosen to be the optimal original medium for optimization. Soluble starch and yeast extract were identified as the optimal carbon and nitrogen source using one-factor-at-a-time method. Response surface methodology was used to optimize medium compositions (soluble starch, yeast extract and inorganic salts). A higher yield of GP-1 was 601.33 µg/L after optimization. The optimal compositions of medium were: soluble starch 13.61 g/L, yeast extract 4.19 g/L, NaCl 3.54 g/L, CaCO3 0.28 g/L, millet, 10 g/L. The yield of GP-1 in a 5 L fermentor using optimized medium was 2.54 mg/L, which is much higher than the result of shake flask. This work will be helpful for the improvement of GP-1 production on a large scale and lay a foundation for developing it to be a novel anti-plant virus agent.

Immunomodulatory activity of macromolecular polysaccharide isolated from Grifola frondosa.

The present study was designed to evaluate the immune-modulating effects of the polysaccharide from Grifola frondosa (GFP) by using mouse peritoneal macrophage and cytoxan (CTX) induced immunosuppression models. Our results from the phagocytotic and mononuclear phagocytic system function assays showed that GFP-A (one component from GFP) stimulated the phagocytosis of the phagocytes. The splenocyte proliferation assay showed that GFP-A acted the effect combing ConA or LPS in splenocyte proliferation. The results showed that GFP-A increased indices of thymus and spleen, the levels of LDH and ACP in the spleen, the mRNA levels of IL-1ß, IL-2, IL-6 and IFN-γ in splenocyte. And GFP-A also significantly increased the expression of CD4(+) and CD8(+) splenic T lymphocytes, which were suppressed by the CTX in peripheral blood. In conclusion, our results indicate that the GFP-A is involved in immunomodulatory effects leading to its modulatory effects on immunosuppression.

Effects of plant stress signal molecules on the production of wilforgine in an endophytic actinomycete isolated from Tripterygium wilfordii Hook.f.

The endophytic actinomycete F4-20 was isolated from Tripterygium wilfordii Hook.f. and was confirmed to produce wilforgine, a secondary metabolite discovered in its host. F4-20 showed a close phylogenetic relationship to Streptomyces species. To seek elicitors that may enhance the production of wilforgine in F4-20, four plant stress molecules were applied to the in vitro liquid cultures. Results showed that methyl jasmonate (MeJA), salicylic acid (SA), and hydrogen peroxide (H2O2) inhibited bacterial growth, whereas glutathione (GSH) treatment significantly increased bacterial growth. The wilforgine contents in the mycelia of F4-20 were reduced by MeJA and GSH but were induced by SA and H2O2. When added in the end of the culture period (7 day), 1 mM SA and 5 mM H2O2 resulted in 69.35 ± 1.71 and 71.80 ± 3.35 µg/g DW of wilforgine production, 1.55 and 1.60 fold to that of control (44.83 ± 1.35 µg/g DW), respectively. Though this improved production was about 6.5 times lower than that of the natural root (454.00 µg/g dry root bark), it provided an alternative method for the production of valuable plant secondary metabolites.

Synthesis, characterization, and antibacterial activity of cross-linked chitosan-glutaraldehyde.

This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR) band at 1559 cm⁻¹, a specific X-ray diffraction peak centered at 2θ = 15°, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug.

Biological Control of Apple Ring Rot on Fruit by Bacillus amyloliquefaciens 9001.

Apple ring rot disease, caused by Botryosphaeria dothidea (Moug. ex. Fr) Ces. et de Not., is one of the most important diseases on apple fruits. In this study, strain 9001 isolated from healthy apple fruits from an infested orchard was evaluated for its biocontrol activity against apple ring rot in vitro and in vivo. Strain 9001 showed obvious antagonistic activity to B. dothidea YL-1 when plated on potato dextrose agar. Soaking healthy apples in the bacterial suspensions of strain 9001 prior to artificial inoculation of fungal pathogen resulted in a dramatic decrease in disease incidence when compared to the control. Moreover, either field application in the growth season or postharvest treatment of apples from infected orchards with bacterial suspensions of strain 9001 resulted in significantly reduced disease incidence within the storage period for 4 months at room temperature. Based on the phylogenetic analysis of 16S rRNA and the gyrA gene, strain 9001 was identified as Bacillus amyloliquefaciens. These results indicated that B. amyloliquefaciens 9001 could be a promising agent in biocontrol of apple ring rot on fruit, which might help to minimize the yield loss of apple fruit during the long postharvest period.

Statistical optimization of process variables for antibiotic activity of Xenorhabdus bovienii.

The production of secondary metabolites with antibiotic properties is a common characteristic to entomopathogenic bacteria Xenorhabdus spp. These metabolites not only have diverse chemical structures but also have a wide range of bioactivities of medicinal and agricultural interests. Culture variables are critical to the production of secondary metabolites of microorganisms. Manipulating culture process variables can promote secondary metabolite biosynthesis and thus facilitate the discovery of novel natural products. This work was conducted to evaluate the effects of five process variables (initial pH, medium volume, rotary speed, temperature, and inoculation volume) on the antibiotic production of Xenorhabdus bovienii YL002 using response surface methodology. A 2(5-1) factorial central composite design was chosen to determine the combined effects of the five variables, and to design a minimum number of experiments. The experimental and predicted antibiotic activity of X. bovienii YL002 was in close agreement. Statistical analysis of the results showed that initial pH, medium volume, rotary speed and temperature had a significant effect (P<0.05) on the antibiotic production of X. bovienii YL002 at their individual level; medium volume and rotary speed showed a significant effect at a combined level and was most significant at an individual level. The maximum antibiotic activity (287.5 U/mL) was achieved at the initial pH of 8.24, medium volume of 54 mL in 250 mL flask, rotary speed of 208 rpm, temperature of 32.0°C and inoculation volume of 13.8%. After optimization, the antibiotic activity was improved by 23.02% as compared with that of unoptimized conditions.

[Experimental study of nimodipine and vascular endothelial growth factor in proliferative retinopathy].

OBJECTIVE: To study the therapeutic effect of the calcium channel antagonist nimodipine on the proliferative retinopathy and it's interaction with vascular endothelial growth factor (VEGF). METHODS: A proliferative retinopathy model (OIR) of newborn Sprague-Dawley (SD) rats was induced by hyperoxia. Different dosages of nimodipine were injected to the rats through retrobulbar or intraperitoneal routes. Both eyeballs of newborn rats were enucleated for performing pathological sections and were studied by immunohistochemical method, in order to count the nuclei of proliferative retinal vessels and to investigate the expression of VEGF in the retina. RESULTS: The number of nuclei of proliferative retinal vessels and the expression of VEGF in non-treatment group increased significantly as compared with normal control group (P < 0.01). Both parameters decreased significantly in high dosage and medium dosage of retrobulbar injection group as compared with the non-treatment group (P < 0.01) and there was no significant decrease in low dosage group (P > 0.05). In each dosage group of intraperitoneal injection, there was a significant decrease of the expression of VEGF (P > 0.01). CONCLUSION: VEGF can induce cell proliferation by activating the calcium channel in cell membrane through which the influx of calcium is increased. The calcium channel antagonist nimodipine can inhibit proliferative retinopathy by blocking the influx of calcium. Nimodipine can inhibit the expression of VEGF at certain degrees.