Objective To develop a rapid,sensitive,specific and accurate quantitative duplex real-time PCR assay for detection of Listeria monocytogenes and Shigella.Methods Two sets of specific primers and probes were selected according to Listeria monocytogencs hly gene and Shigella ipaH gene.The target hly and iPaH fragments were amplified by PCR,and used to construct recombinant pGEM-T-hly and pGEM-T-ipaH respectively.The two recombinant circular plasmid DNAs were linearized with EcoR I that did not cut within the target DNA fragment.The ten-fold dilutions of plasmid were subjected to the standard quantitation curve in duplex real-time PCR assay.Various genomic DNAs of Listeria innocua,Listeria weshimeri,Salmonella,Staphylococcus aureus,Bacillus subtilis,Escherichia coli and Proteus were used as negative controls to confirm the specificity of duplex real-time PCR assay.The assay was also used to detect Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk.Results The recombinant plasmids were constructed successfully,hly probe(rAM and TAMRA double labelled)and ipaH probe (HEX and TAMRA double labelled)were used to develop an optimized PCR successfuliv.Conclusion The selected primers and probes showed high specificity for these two target bacteria,the linear range of the assay was good(105-101 copies/μl,R2≥0.998)and sensitivity Was 10 copies/PCR.Following a DNA extraction method which combined EZ Spin Colum Genomic DNA Isolation Kit(BBI)/Phenol-chloroform,the sensitivity of assay Was 102CFU/ml for both Listeria monocytogenes and Shigella in artificially contaminated sterilized skim milk,which equivalents to 10 CFU/PCR.

Objective To explore the protective mechanism of tea polyphenols (TPs) ion oxidative stress damage of the rat articular cartilage tissue caused by brick-tea fluorosis. Methods One hundred and twenty wistar male rats were randomly divided into 6 groups according to body mass: fluoride group with drinking water containing 100.00 mg/L F-, fluoride plus TPs group treated with 100.00 mg/L F- and 10.0 g/L TPs, fluoride plus aluminum group fed with 100.00 mg/L F- and 200.00 mg/L Al3+, fluoride plus aluminium and TPs group treated with 100.00 mg/L F-,200.O0 mg/L Al3+ and 10.0 g/L TPs;brick-tea group treated with drinking water containing 100.00 mg/L F-,215.00 mg/L Al3+ and 9.2 g/L TPs, which was steeped by the brick-tea;control group treated with tap water. The animals were bred for three months and then sacrificed. The level of SOD,T-AOC and MDA in blood serum were detected,also the level of NO and cytokine IL-1β and IL-6, the expression of iNOS mRNA and protein in articular cartilage were respectively analyzed by RT-PCR and immunohistochemistry. Results Blood serum SOD level in the fluoride plus aluminum and TPs group[(664.009 ± 29.589)kU/L] was higher compared with that in the fluoride group[(625.328 ± 27.199)kU/L], fluoride plus aluminum group[(652.282±13.926)kU/L], although no statistically significant differences was found(P > 0.05) ;blood serum T-AOC level of the fluoride plus TPs, fluoride plus aluminum and TPs group, brick tea group[(10.874 ± 0.721), (11.871 ± 0.941), (10.380 ± 2.747)kU/L] was higher compared with fluoride group, fluoride plus aluminum group [(8.849 ± 1.887), (8.210 ± 1.740)kU/L], the differences all being statistically significant(P < 0.05) ;blood serum MDA level in the fluoride plus aluminum and TPs group[(3.235 ± 0.446)μmol/L] had significances compared with fluoride group, fluoride plus aluminum group [(3.889 ± 0.387), (4.580 ± 0.474)μmol/L, all P < 0.05)];blood serum NO level in fluoride plus Tps group, fluoride plus aluminum and TPs group, brick-tea group[(23.278 ± 2.386), (20.643 ± 2.623), (24.367 ± 6.072) μmol/L] had tatistical differences compared with fluoride group, fluoride plus aluminum group[(32.962 ± 8.268), (34.909 ± 6.288)μmol/L, all P < 0.05];blood serum IL-1β level of fluoride group, fluoride plus aluminum, fluoride plus Tps, fluoride plus aluminum and TPs group and brick-tea group [(4.728 ± 0.297), (4.412 ± 0.229), (4.432 ± 0.285), (4.516 ± 0.351), (4.614 ±0.2270)n/L] did not have inter-group differences (F = 2.314,P > 0.05);the blood serum IL-6 level of fluoride plus aluminum and TPs group, brick-tea group[(7.231 ± 0.596), (7.325 ± 0.290)ng/L] had statistical differences compared with fluoride plus aluminum[(8.256 ± 0.635)ng/L, P < 0.05]. The iNOS mRNA correspondent expression content of fluoride plus Tps group, fluoride plus aluminum and TPs group, brick-tea group(0.482 ± 0.021,0.447±0.021,0.491 ± 0.022) had statistical differences compared with fluoride group, fluoride plus aluminum group (0.562 ± 0.025,0.591 ± 0.020, all P < 0.05). Cells with positive iNOS protein expression of control group were mainly distributed at the surface layer of joint, while the cells of experiment groups were distributed both at the surface layer and the intermediate layer. Conclusions Tea polyphenols could alleviate oxidative stress damage on the articular cartilage, exerting protection against brick-tea fluorosis on rats through cleaning up free radicals, elevating total anti-oxidation capability, diminishing the generation of lipid peroxide.

Adenoma, Villous ; metabolism ; pathology ; surgery ; Aged ; Diagnosis, Differential ; Female ; Humans ; Keratin-20 ; metabolism ; Keratin-7 ; metabolism ; Mullerian Ducts ; pathology ; Papilloma ; pathology ; Vaginal Neoplasms ; metabolism ; pathology ; surgery

Objective To provide a basis for preventive strategies to national drinking-water-borne endemic arsenicosis through mastering the implementing progress of preventive measures and observing the dynamic changes.Methods Surveillances were carried out according to the provisions and requirements of The Surveillance Project for National Drinking-Water-Borne Endemic Arsenicosis(Trial).Total of 11 provinces(autonomous regions) and Xinjiang Production and Construction Corps were selected as the surveillance provinces (autonomous regions).Endemic arsenicosis villages with exposed population over 100 persons were chosen as monitoring villages in each province,81 villages in 2010 and 89 villages in 2011 were selected.Potential endemic arsenicosis villages with exposed population over 100 persons were included; 26 villages in 2010 and 19 villages in 2011 were selected.The operation of water-improving projects was investigated,the arsenic content in water from resident house was tested in potential endemic arsenicosis villages and the prevalence of endemic arsenicosis based on the residents who lived in monitoring villages was surveyed:Results ①Total of 225 water-improving projects in 45 counties were monitored,1349 villages were covered and 72.66 million persons were benefited in 2010.Total of 233 waterimproving projects in 48 counties were monitored,1576 villages were covered and 84.61 million persons were benefited in 2011.②)Total of 107 villages with high level of water arsenic were investigated and 81 villages had improved the water quality in these villages in 2010.The water-improving projects running normally reached 90.12％(73/81),intermittent operation rate was 9.88％ (8/81) and without abandoned projects.The projects with qualified water reached 86.42％ (70/81).Total of 108 villages with high level of water arsenic were investigated and 89 villages with water improved in 2011.Normally operated projects reached 86.52％ (77/89),intermittent operation rate was 11.24％ (10/89)and abandoned projects was 2.25％ (2/89).The projects with qualified water arsenic level reached 82.02％(73/89).In addition,26 villages without water-improvement were investigated in 2010,and the families with high level of water arsenic reached 66.01％(371/562).Total of 19 villages were surveyed in 2011,and the families with high level of arsenic reached 54.99％(204/371).③Total of 23 964 persons were examined in villages with improved water in 2010,the detection rate of patients with endemic arsenicosis was 4.43％ (1061/23 964),3964 persons were examined in the villages without water-improvement and the detection rate was 5.98％(237/3964),two new cases were diagnosed.Total of 25 225 persons were examined in villages with waterimproved,the detection rate was 4.68％(1181/25 225),3145 persons were examined in the villages without waterimprovement,and the detection rate was 2.26％(71/3145) in 2011,none new case was detected.Conclusions It is not optimistic about the operating status and quality of water-improving projects.The prevalence in water-improved villages remains higher than that in water-unimproved villages.The long-term mechanism of surveillance should be established and perfected as soon as possible,and the management and maintenance of water-improving projects also should be strengthened.

Objective To investigate the disease situation of adult carotid atherosclerosis in water-related endemic fluorosis areas in Heilongjiang province in 2008 so as to explore the relationship between water-related endemic fluorosis and incidence of carotid atherosclerosis disease. Methods A total of 266 participants over the age of 40 from four villages in Zhaozhou county with water fluoride ≥ 1.0 mg/L in a fluorosis area and 283 residents over the age of 40 from four villages in Tailai county with water fluoride ＜ 1.0 mg/L in a nonfluorosis area were investigated. A portable-type B mode color ultrasound was used to examine the left carotid artery of all participants.The carotid atherosclerosis was diagnosed and graded through the ultrasonograms. Results The prevalence rates of carotid atherosclerosis in the 4 fluorosis villages(Xinfeng, Taipingshan, Baochan and Houzheng villages) were 47.3% (35/74), 63.5%(40/63), 73.3%(33/45) and 60.7%(51/84), respectively, and in the 4 nonfluorosis villages(Hala,Qianxing, Sanjia and Ailin villages) were 32.7% (17/52), 32.9% (24/73), 39.2% (31/79) and 30.4% (24/79),respectively. The prevalence rates of carotid atherosclerosis in every villages standardized by age were 47.3% ,63.5%,73.3% ,60.7% and 34.7% ,36.3% ,43.0% ,41.3%, respectively. Statistic method used was Wilcoxon two sample test and the differences were significant(T = 26, P ＜ 0.05 ). Total carotid atherosclerosis positive rate standardized by age in the 4 fluorosis villages was significantly higher than that in the 4 nonfluorosis villages[57.5%(153/266) ,37.8% (107/283), x2 = 21.36, P ＜ 0.01 ]. After standardized by age, the severity of carotid atherosclerosis was significantly different between fluorosis villages and nonfluorosis villages (x2 = 36.15, P ＜ 0.01 ). Conclusion The prevalence rate of carotid atherosclerosis in endemic fluorosis area is higher than that in nonfluorosis area.

Objective To observe the influence of fluoride and aluminum on the expression of matrix metalloproteinase-13(MMP-13) in rat articular chondrocytes. Methods Original generation chondrocytes of rats was cultured and divided into fluoride group, aluminum group, fluoride plus aluminum group and control group. NaF and A1C13 at concentrations of 1 mmol/L and 2 mmol/L were administered to intoxicate the cells for 24, 48, 72 h respectively. Cells were extracted to undergo reverse transcription the polymerase chain reaction(RT-PCR) at different times to observe mRNA expression of MMP-13, and protein expression was detected by Western-blot. Results In 24 h, the content of MMP-13 mRNA in fluoride group(0.830±0.043), aluminum group(1.279±0.060) and fluoride plus aluminum group(0.983±0.028) was higher than that in the control group(0.707±0.026, P<0.05), and relative expression of MMP-13 mRNA in aluminum group was the highest. In 48 h, the content of MMP-13 mRNA in fluoride group (0.964±0.180), aluminum group (1.333±0.105) and fluoride plus aluminum group (0.915±0.137) was higher than that in the control group(0.660±0.055, P<0.05), and the relative expression in aluminum group was the highest. In 72 h, the content of MMP-13 mRNA in fluoride group(0.866±0.115), aluminum group(0.846±0.089) and fluoride plus aluminum group(0.967±0.196) had no statistical significance(P>0.05) compared with the control group(0.809±0.179). In 24 h, the content of MMP-13 protein in fluoride group(1.050±0.084), aluminum group(1.010±0.113) and fluoride plus aluminum group(0.977±0.202) had no statistical significance(P>0.05) compared with the control group(0.860±0.038). In 48 h, the content of MMP-13 protein in fluoride group(0.671±0.020), aluminum group(1.134±0.094) and fluoride plus aluminum group (0.923±0.087) was higher than that in the control group (0.647±0.025, P<0.05), but no significant difference being observed between groups (P>0.05). In 72 h, the content of MMP-13 protein in fluoride group(0.672±0.022), aluminum group(1.088±0.072) and fluoride plus aluminum group(0.772±0.030) was higher than that in the control group(0.577±0.026, P<0.05). It was the highest in the aluminum group, the intra-group difference had statistical significance(P<0.05). Conclusions Fluoride and aluminum damage chondrocytes to some extent, toxicity of aluminum itself is greater than fluoride and fluoride plus aluminum. Abnormal expression of MMP-13 can be observed in the chondrocyte damage process induced by fluoride and aluminum.

ObjectiveTo investigate the protective effect of all-trans retinoic acid (ATRA) on injury of human immortalized hepatocytes (HHL-5 cells ) induced by sodium arsenite and possible mechanisms.Methods After cultured for 48 h,HHL-5 cells were divided into four groups:normal group,ATRA group,sodium arsenite group and ATRA + sodium arsenite group.HHL-5 cell viability was tested by using cell proliferation experiment (WST).Superoxide dismutase(SOD),glutathione peroxidase(GSH-Px) activity,malondialdehyde(MDA) content,and aspartate aminottransferase (AST) activity in each group were determined by biochemical method.The microstructure of HHL-5 cells in each group was observed under transmission electron microscopy.ResultsHHL-5 cell viability(0.57 ± 0.02) of sodium arsenite group was compared with that of normal group(0.70 ± 0.01 ),the difference was statistically significant(P ＜ 0.05).Levels of SOD,GSH-Px,MDA and AST[ (153.84 ± 2.35),(0.08 ±0.02)U/mg Prot,(4.15 ± 0.50)nmol/mg Prot,(265.43 ± 4.62) × 103 U/L] of sodium arsenite group were compared with that of normal group[(237.41 ± 18.30),(0.93 ± 0.02)U/mg Prot,(2.26 ± 0.40)nmol/mg Prot,(177 ± 9.85) ×103 U/L],and the difference was statistically significant (all P ＜ 0.05).HHL-5 cell viability (0.65 ± 0.04) of ATRA + sodium arsenite group was compared with that of sodium arsenite group, and the difference was statistically significant (P ＜ 0.05).Levels of SOD,GSH-Px,MDA and AST[ (286.85 ± 3.39),(0.56 ± 0.09)U/mg Prot,(3.36 ± 0.37)nmol/mg Prot, (220.02 ± 1.07) × 103 U/L] of ATRA+ sodium arsenite group were compared with that of sodium arsenite group,the difference was statistically significant(all P ＜ 0.05).Compared with normal group and ATRA group,the surface microvilli of HHL-5 cells of sodium arsenite group decreased,double-membrane structure was unclear,vacuolar degeneration was seen in the cytoplasm,and glycogen was aggregated.The damage level of ATRA + sodium arsenite group was decreased.ConclusionsATRA plays a protective role through increasing intracellular antioxidant enzyme activity of HHL-5 cells,removal or reduction of oxygen free radicals produced by sodium arsenite.

Objective To observe the influences of different doses of sodium arsenite on mRNA transcription of keratinizing related and nuclear factor E2-related factor 2(Nrf2) genes in HaCaT cells.Methods Cell proliferation was evaluated by Cell Counting Kit-8(CCK-8) assay after the HaCaT cells were exposed to 0.00,3.13,6.25,12.50,25.00,50.00,75.00,100.00 μ mol/L sodium arsenite for 48 h,respectively.Based on the previous results of cell proliferation,0.00(control),6.25,12.50,and 25.00 μmol/L of sodium arsenite were selected to treat HaCaT cells for 48 h,respectively.The mRNA expression of keratin 1,keratin 10,involucrin,loricrin and Nrf2 were detected by real-time fluorescent quantitative PCR.ResultsCompared with the control group (100.05％),HaCaT cell proliferation rates(83.06％,51.04％,39.52％,24.51％,16.99％ and 9.04％) were significantly lower in 6.25,12.50,25.00,50.00,75.00 and 100.00 μ mol/L of sodium arsenite groups and the 50％ inhibiting concentration was 12.38 μmol/L.Compared with the control group( 1.06 ± 0.28,1.00 ± 0.12,1.00 ± 0.08),the mRNA expression of keratin 1,involucrin and loricrin (0.08 ± 0.04,0.13 ± 0.12,0.05 ± 0.03;0.47 ± 0.11,0.21 ± 0.09,0.10 ± 0.15; 0.50 ± 0.27,0.31 ± 0.10,0.57 ± 0.23) were significantly decreased(all P ＜ 0.05) in HaCaT cells treated with 6.25,12.50,25.00 μmol/L sodium arsenite,respectively.But keratin 10 mRNA expression showed a rise trend and the 6.25 μmoL/L sodium arsenite group (1.83 ± 0.45) was significantly higher than that of the control( 1.07 ± 0.14,P ＜ 0.05 ).The Nrf2 mRNA expressions of HaCaT cells in 12.50,25.00 μmol/L sodium arsenite groups(0.13 ± 0.07,0.69 ± 0.33) were significantly lower than that of the control ( 1.00 ± 0.09,all P ＜ 0.05 ).ConclusionsThe cellular proliferation and keratinization are decreased when HaCaT cells are exposed to sodium arsenite,which may be regulated by lowering Nrf2 mRNA transcription.

Objective To establish a new determination method for the measuring of alkaline phosphatase activity (ALP) with p-acetyl phenyl phosphace (PAP-PNa_2) as substrate.Methods With the help of Vital semiautomatic analyzer,researched a continuous-monitoring procedure and set up experimental parameters.Results When using this assay,the wavelength of PAP's absorption was 325 nm and the Km of ALP was 0.376 mmol/L.The molecular extinction coefficient of PAP at 340 nm was 23 390 L?mol~(-1)? cm~(-1) and the concentration of citrate buffer was 0.438 mol/L.During the process,we found that the optimum pH of enzyme was 10.4,and the concentration of substrate was 5.0 mmol/L.The time of linear reaction was 900 seconds,and the linear range was 0-1 110 U/L.Serum total ALP were 63.1-118.3 U/ L(male) and 52.5-89.0 U/L(female),based on results from 60 heath adults.Conclusions The method is practical in its repetition and convenience,saves time and is not liable to be affected by bilirubin in serum.It is especially suited to the use of automatic analyzers.

Objectives To screen the differentially expressed proteins in serum of population chronically exposed to arsenic in drinking water,thus to provide candidate protein biomarkers for arsenic exposure and arsenicosis.Methods Subjects were selected from the drinking water type of endemic arsenicosis areas in Shanxi province,China.Demographic characteristics,history of arsenic exposure,cigarette smoking,alcohol drinking,health and other information were collected using questionnaire.The subjects were divided into low-arsenic group (with arsenic in drinking water ＜ 10 μg/L),medium-arsenic group( 10 - 50 μg/L),high-arsenic group( ＞ 50 μg/L),and arsenicosis group(the drinking water with arsenic ＞ 50 μg/L was replaced by low arsenic water ＜ 10 μg/L).The number of cases in each group was 30.The arsenicosis patients were diagnosed according to “Standard of Diagnosis for Endemic Arsenism” (WS/T 211-2001 ).With the principle of informed consent,blood samples were collected.Differentially expressed serum proteins of different arsenic exposure groups and arsenicosis group were screened by two-dimensional differential gel electrophoresis(2-D DIGE),and further identified by mass spectrometry (MS).Results An average of (1299 ± 167) protein spots were identified in 6 gel images and 688 protein spots were discovered repeatedly in at least 5 gels.There were 33 protein spots differentially expressed among low-,medium- and high-arsenic groups P ＜ 0.01).Fifty four protein spots were significantly different among low-,medium-,high-arsenic exposure groups and arsenicosis group(P ＜ 0.01 ).Twenty five protein spots were selected for MS analysis,and13 protein spots were identified.Compared with low-arsenic group,the expressions of apolipoprotein A-Ⅳ,retinol binding protein,and estrogen receptor hypothalamic isoform in medium- and higharsenic exposure groups were down regulated,and the expressions of component 4A and 4B were up regulated.Compared with low-,medium- and high-arsenic groups,the expressions of beta-2-glycoprotein Ⅰ,Keratin 1,hemopexin,complement C1r subcomponent,and ficolin-3 in arsenicosis group were down regulated,and the expressions of pigment epithelial-differentiating factor,alpha-1-microglobulin and carboxypeptidase N catalytic chain were up regulated.Conclusions Chronic arsenic exposure can significantly change population's serum protein expression.Differentially expressed proteins in arsenicosis patients will not decline with the decline of arsenic in a short term.Whether or not the differentially expressed proteins identified in this study can be used as biomarkers for arsenic exposure and arsenicosis needs to be further verified.