Nitrate Quantification in Fresh Vegetables in Shanghai: Its Dietary Risks and Preventive Measures.

To investigate nitrate and nitrite content in fresh vegetables, 264 samples were randomly collected in the farmers' markets in Shanghai, Southeast China. The results indicate that 25.0% of the fresh vegetables were critically or more contaminated by nitrate [>1440 mg/kg FW (Fresh weight)]. Generally, leafy vegetables were more highly enriched in nitrate than root-tuber and fruit vegetables. About 22.6% of the leafy vegetables had a nitrate content exceeding the limit for edible permission (>3000 mg/kg FW). Nitrite content in the fresh vegetables was all within the safe level (<1 mg/kg FW). It was estimated that the daily nitrate intake through eating vegetables in Shanghai exceeded the WHO/FAO allowable limit. The field experiment indicated that the hyper-accumulation of nitrate and nitrite in the vegetables was mainly attributed to the excessive application of chemical fertilizers. The maxima of nitrate and nitrite in the vegetables were attained one week after applying chemical fertilizer, and thus they cannot be picked for dietary use. Applying organic manure can effectively lower the risk of nitrate and nitrite contamination in vegetables. The old leaves and leaf petioles were more easily enriched in nitrate due to their weaker metabolic activity. Vegetables with high nitrate content had a high risk of nitrite toxicity during storage due to the biological conversion of nitrate into nitrite, which is easily triggered by suitable temperature and mechanical damage processing. Therefore, fresh vegetables should be stored by rapid cooling and in undamaged forms to prevent nitrite accumulation.

[Ammonia Oxidation in a Neutral Purple Soil Measured by the 13C-DNA-SIP Method].

Increasing evidence suggests that ammonia oxidation in acidic soils is primarily catalyzed by ammonia-oxidizing archaea (AOA), while ammonia-oxidizing bacteria (AOB) drive ammonia oxidation in neutral and alkaline soils in which AOA overwhelmingly outnumber AOB. Therefore, neutral purple soil with a pH of 7.2 was selected to study the composition of the active ammoxidation microbial community with a stable isotope nucleic acid probe technique combined with cloning sequencing. Results showed that the nitrification rate was 9.68 mg·(kg·d)-1, and AOA and AOB were abundant in neutral purple soils. By using DNA-based stable isotope probing (SIP), we gathered strong evidence of archaeal ammonia oxidation by AOA and AOB. Phylogenetic analysis indicated that the Nitrosospira Cluster 3a.1 AOB was dominant in terms of quantity at 0 days, and the Nitrosospira Cluster 3a.2 only accounted for a small part. After 56 days of cultivation, the Nitrosospira Cluster 3a.2 replaced the Nitrosospira Cluster 3a.1 as the active AOB that dominated ammonia oxidation. The AOA that predominated quantitatively at day 0 was Nitrososphaera Subcluster 9, but after cultivation this became Nitrososphaera Subcluster 3.2/3.3. Thus, the community structure of AOA and AOB changed. Active autotrophic nitrification was found in this neutral purple soil. Sequencing analysis of the 13C-labeled DNA provided robust evidence that both archaea and bacteria played important roles in the nitrification and not all ammonia oxidizers in native soil were active in the nitrification. Phylogenetic analysis clearly showed that the dominant active archaea and bacteria during the incubation were affiliated with Nitrososphaera Subcluster 3.2/3.3 within the soil group 1.1b lineage and Nitrosospira Cluster 3a.2, respectively, which were different from the dominant ammonia oxidizers at the beginning of the incubation. These results suggest that the community structure of ammonia oxidizers can shift quickly upon changes in the substrate availability in soils.

[Construction of biofilm formation related mutants in Vibrio parahaemolyticus].

OBJECTIVE: To construct the mutants of biofilm related genes in Vibrio parahaemolyticus and confirm the mutants. METHODS: The homologous upstream and downstream flanking fragments of target gene were amplified by using PCR, and the fusion homologous fragment was amplified by using the two flanking fragments as template. Then the fusion homologous fragment was digested by restriction enzyme and cloned into suicide plasmid pDS132. The recombinant plasmid was transferred into Vibrio parahaemolyticus RIMD 2210633 through conjugation. The mutants were screened and identified by PCR and the phenotype of one mutant was analyzed in order to verify that the mutants were constructed successfully. RESULTS: Six recombinant plasmids carrying the fusion homologous fragments of genes vbfR, crp, hns, swrZ, swrT and cpsR respectively were constructed and identified by PCR. The amplification products of 1190, 1128, 1136, 953, 1242 and 1112 bp were obtained respectively. The six mutants (ΔvbfR, Δcrp, Δhns, ΔswrZ, ΔswrT and ΔcpsR) were constructed using recombinant plasmids. Verified by PCR, the size of amplification products of mutants (1190, 1128, 1136, 953, 1242 and 1112 bp respectively) was less (610, 739, 421, 542, 427 and 1367 bp respectively) than the corresponding positive control. Meanwhile, none of the products was amplified using the primers locating on the target gene. One mutant Δhns was selected to test the ability of biofilm formation. The result showed that the ability of biofilm formation of mutant Δhns was increased compared with the wild type. CONCLUSION: Six mutants of biofilm related genes in Vibrio parahaemolyticus were constructed and tested by molecular and phenotype experiment to confirm that the mutants were constructed successfully.

Affects of mining activities on Cd pollution to the paddy soils and rice grain in Hunan province, Central South China.

Located in Central South China, Hunan province is rich in mineral resources. To study the influence of mining on Cd pollution to local agricultural eco-system, the paddy soils and rice grain of Y county in northern Hunan province were intensively monitored. The results were as follows: (1) Total Cd (T-Cd) content in the soils of the county ranges from 0.13 to 6.02 mg kg(-1), with a mean of 0.64 mg kg(-1), of which 57.5% exceed the allowable limit specified by the China Soil Environmental Quality Standards. T-Cd in the soils varies largely, with the coefficient of variation reaching 146.4%. The spatial distribution of T-Cd in the soils quite matches with that of mining and industries. The content of HCl-extractable Cd (HCl-Cd) in the soils ranges from 0.02 to 2.17 mg kg(-1), with a mean of 0.24 mg kg(-1). A significant positive correlation exists between T-Cd and HCl-Cd in the soils (r = 0.770, ρ < 0.01). (2) Cd content in the rice produced in Y county ranges from 0.01 to 2.77 mg kg(-1), with a mean of 0.46 mg kg(-1). The rate of rice with Cd exceeding the allowable limit specified by the Chinese Grain Security Standards reaches 59.6%; that with Cd exceeding 1 mg kg(-1), called as "Cd rice," reaches 11.1%. (3) Cd content in the rice of Y county is positively significantly correlated with HCl-Cd (r = 0.177, ρ < 0.05) but not significantly with T-Cd in the soils (r = 0.091, ρ > 0.05), which suggests that the amount of Cd accumulating in the rice is more affected by its availability in the soils, rather than the total content. (4) The dietary intake of Cd via rice consumption in Y county is estimated to be 179.9 µg day(-1) person(-1) on average, which is far beyond the allowable limit specified by FAO/WHO and the target hazard quotients of Cd much higher than 1, suggesting the high risk on human health from Cd exposure.

[Research on the carbon content of coal by LIBS].

A method is introduced about quantitative analysis of carbon in coal by LIBS (laser-induced breakdown spectroscopy) in the present paper, and it introduces data optimization technology and method based on spectral data integration, normalization and data screening processing to overcome the poor quality of precision in the analysis because of laser source energy fluctuation, self-absorption, sample surface roughness etc. It is showed that the standard deviation (SD) is less than 1.6% using the method to analyze C element content in coal, and this method also can be used for other element analysis for coal.

[Analysis of software for identifying spectral line of laser-induced breakdown spectroscopy based on LabVIEW].

Self-designed identifying software for LIBS spectral line was introduced. Being integrated with LabVIEW, the soft ware can smooth spectral lines and pick peaks. The second difference and threshold methods were employed. Characteristic spectrum of several elements matches the NIST database, and realizes automatic spectral line identification and qualitative analysis of the basic composition of sample. This software can analyze spectrum handily and rapidly. It will be a useful tool for LIBS.

[Research on parameters optimization of laser-induced breakdown spectroscopy based experimental device].

For a better application of laser-induced breakdown spectroscopy (LIBS) to coal quality analysis, it is necessary to optimize the key parameters of the experimental LIBS-based device. The relationships between the key parameters and the signal-to-noise ratios (S/N) of the elemental emission lines in the plasma spectrum of the pulverized coal were studied, according to which the optimal parameters can be selected. Experimental results indicate that the optimal settings for our LIBS-based device are laser pulse energy = 120 mJ x Pulse(-1), delay time of spectrometer = 200 ns, laser focal point be located 3-5 mm underneath the sample surface, rotation speed of sample cell = 2.7 rev x min(-1), a narrow-band filter with center frequency of 1 064 nm and a diaphragm with center hole diameter of 1.5 mm be placed in the path of the laser beam. Quantitative analysis results of pulverized coal show that, by using the optimal LIBS-based device, the standard deviation (SD) of C has been reduced from 6.7% to 1.6%, while the relative standard deviation (RSD) of other trace elements has been reduced from 28% to 10%. As a result, th accuracy has been improved greatly.

[Establishment of stable subline of K562 cells expressing human leucocyte antigen a1101].

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.

Lipopolysaccharide initiates a bypass feedback loop of epidermal growth factor receptor signaling by HPS70-induced COX-2 in H22 hepatocarcinoma cells.

LPS can induce TACE upregulation via signaling from TLR4-derived EGFR activation in tumor cells. The regulation and activity of TACE have been investigated with the observation that gene expression is upregulated in response to LPS followed by EGFR activation, however, the process remains poorly understood. In this study, we examined the effects of LPS on H22 hepatocarcinoma cells that displayed constitutively active TLR4 expression. Upon TLR4 shRNA transfection into H22 cells, HSP70 expression significantly increased. However, LPS induced early phosphorylation of EGFR in H22 cells, which reached maximum levels within 30 min. Inhibition of TLR4 in H22 cells resulted in a significant rise in both EGFR phosphorylation and TACE upregulation 24 h after exposure to LPS. Exogenous HSP70 also induced rapid phosphorylation of EGFR, upregulated the expression of COX-2 via a signaling pathway that involved TACE-dependent TGF-α release. Furthermore, inhibition of EGFR activation and reduction of COX-2 expression by COX-2 inhibitor prevented HSP70-induced cell invasion in vitro. These findings demonstrate that the biological importance of HSP70/COX-2 is crucial to the second, but not the first, phase of EGFR phosphorylation in tumor cells. The growth of tumor cells by inserting shRNA plasmid TLR4 combination with COX-2 inhibitor could be effectively reduced in LPS stimulation. We concluded that LPS triggered a bypass feedback loop of EGFR activation and involved HSP70/COX-2 in H22 cells by inhibition of TLR4 and that EGFR phosphorylation is implicated in tumor growth by LPS stimulation.

[The expressions and clinical significance of IGFBP-2, -3 in both serum and tumor tissues in patients with epithelial ovarian cancer].

OBJECTIVE: To explore the serum levels of IGFBP-2, -3 and their proper roles in the regulation of IGF-II bioavailability in patients with ovarian tumor, and to investigate the correlation between the expressions of IGFBP-2 and IGFBP-3 in ovarian tumor tissues and related clinicopathological characteristics. METHODS: Serum levels of IGFBP-2, -3 and big/mature IGF-II were measured by Western ligand blot (WLB) and Western blot (WB) in patients with ovarian tumor (10 cases of benign tumor, 6 cases of borderline tumor and 10 cases of malignant tumor) and 10 cases of normal control. The expressions of IGFBP-2 and IGFBP-3 were examined in 39 specimens of ovarian tumor (8 cases of benign tumor, 8 cases of borderline tumor and 23 cases of malignant tumor) and 4 cases of normal ovarian tissues by immunohistochemical staining. RESULTS: The serum levels of both big and mature IGF-II in epithelial ovarian cancer (ovarian cancer) patients were significantly decreased compared with those of normal control and benign and borderline tumor (P<0.001 or P<0.01). The increased serum level of IGFBP-2 and decreased IGFBP-3 level were observed in patients with malignant ovarian tumors by comparing with those of patients with normal controls, benign and borderline tumor (P<0.001 or P<0.01). The expression of IGFBP-2 was significantly higher in malignant ovarian tumor tissues than those in normal control and benign ovarian tumors tissues (P<0.0001, P<0.001, and the expression of IGFBP-3 decreased significantly in lower differentiated ovarian cancer tissues compared with that in high and moderate differentiated ovarian cancer tissues (P<0. 05). CONCLUION: IGFBP-2 predominantly presents in the circulation of malignant patients in contrast to IGFBP-3, which may result in altered bioavailability of IGF-II in ovarian cancer, leading to the progress of tumor. The serum levels of both IGFBP-2 and IGFBP-3 and their expressions in tumor tissues are correlated with the clinicopathological characteristics of ovarian cancer patients. Our findings suggest that the presence of new mechanisms in the regulation of IGF-II bioavailability, and provide the evidence for the possibility to use IGFBP-2/IGFBP-3 as biological markers in diagnosis and prognosis of ovarian cancer.