Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.

Objective To get the gene encoding extracellular domains of gB protein of B virus and analyze its expression in the eukaryocyte cell.Methods synthesizing gene fragment encoding extracellular domains of gB protein of B virus was by using synthesis gene, then digested with the restriction endonucleases BamHⅠand NotⅠand inserted into eukaryotic expressing vector pEGFP-N3.pEGFP-N3-GB合 was transfected into 293 cells.After protein extraction, the expression of gene was detcted by western blotting, and the cellular localization of the gene was analyzed by immunofluorescence and laser scanning confocal microscopy.Results pEGFP-N3-GB合were expressed in 293 cells and on the cell membrane.Conclusion eukaryotic expressing system can produce specific antigen recombination protein of B virus gB protein and express on the cell membrane.

Objective To detect the modification levels of H2AKll9 ubiquitination (H2AK119ub) and H2BK120ub,and to analyze the relationship between the levels of H2AK119ub,H2BK120ub and arsenic exposure.Methods A cross-sectional study was conducted in typical areas of drinking water type of endemic arsenicosis in Shanxi and Jilin provinces.Totally 281 residents who had drank local water for more 10 years were enrolled in this study,these participants were divided into control group (water arsenic content ＜ 0.01 mg/L),low arsenic exposure group (water arsenic content ranged 0.01-0.05 mg/L),medium arsenic exposure group (water arsenic content ranged ＞ 0.05-0.10 mg/L) and high arsenic exposure group (water arsenic content ＞ 0.10 mg/L).Among them,including 60 subjects in control group (20 males and 40 females),61 subjects in low arsenic exposure group (27 males and 34 females),50 subjects in medium arsenic exposure group (17 males and 33 females),and 110 subjects in high arsenic exposure group (40 males and 70 females).Drinking water and urine samples were collected and the arsenic content was detected by the method of atomic fluorescence spectrometry.After extracting leukocytes histone from the peripheral venous blood that collected from the subjects,the levels of H2AK119ub and H2BK120ub were detected by dot blotting.The levels of water arsenic,urinary arsenic,water arsenic accumulative intake,H2AK119ub and H2BK120ub were expressed as medium and quartile [M (P25,P75)].Results Age,body mass index (BMI),gender,smoking and alcohol drinking between control group and water arsenic exposure groups had no statistical differences (x2 =3.780,3.572,1.938,4.937,6.025,all P ＞ 0.05).Compared the contents of water arsenic [0.005 (0.003,0.006),0.024 (0.017,0.037),0.076 (0.057,0.084),0.150 (0.124,0.185) mg/L],the contents of urinary arsenic [0.011 (0.006,0.017),0.018 (0.004,0.072),0.061 (0.032,0.124),0.134 (0.069,0.223) mg/L],the water arsenic accumulative intake [0.342 (0.248,0.477),1.641 (1.012,2.324),5.273 (3.690,7.036),7.716 (5.608,12.053) mg] among the control,low,medium and high arsenic exposure groups,the differences were statistically significant (Hc =256.041,88.615,218.610,all P ＜ 0.01).Compared the levels of H2AK119ub [1.231 (0.856,1.817),1.244 (0.792,1.884),1.376 (0.743,1.981),1.390 (0.906,2.045)],H2BK120ub [0.350 (0.186,0.589),0.363 (0.152,0.678),0.428 (0.134,0.788),0.276 (0.146,0.453)] in human peripheral blood leukocytes among control,low,medium and high arsenic exposuregroups,the differences were not statistically significant (Hc =2.130,4.330,all P ＞ 0.05).There were no correlations between H2AK119ub and water arsenic content,water arsenic accumulative intake (r =0.104,-0.008,all P ＞ 0.05);there was a positive correlation between H2AK119ub and urinary arsenic content (r =0.166,P ＜ 0.05).There were negative correlations between H2BK120ub and water arsenic content,water arsenic accumulative intake (r =-0.183,-0.159,all P ＜ 0.05);there was no correlation between H2BK120ub and urinary arsenic content (r =-0.101,P ＞ 0.05).There was a negative correlation between H2AK119ub and H2BK120ub (r =-0.127,P ＜ 0.05).Conclusion External exposure to arsenic may change the levels of H2BK120ub in human peripheral blood leukocytes.

Objective To investigate the relationship between single nucleotide polymorphism(SNP)of the peroxisome proliferator activated receptor γ (PPARγ) gene Rs1801282 and brick-tea type fluorosis. Methods From 2012 to 2013, this cross-sectional study was performed in 16 endemic fluorosis areas of brick-tea type in Inner Mongolia Autonomous Region,Qinghai and Xinjiang Uygur Autonomous Region of China,to select adults>18 years old as subjects, who were diagnosed as skeletal fluorosis by X-ray. All of the subjects filled in demography survey questionnaire; the survey contents included general characteristic s, and average daily brick tea intake. Drinking tea samples and urine samples of each subject were collected, and fluoride content of urine and brick-tea was determined via the ion selective electrode method (WS/T 89-2006). X-ray scintigraphy was used to diagnose skeletal fluorosis, according to the "Diagnostic Criteria of Endemic Skeletal Fluorosis" (WS/T 192-2007); the subjects were divided into skeletal fluorosis group (case group) and non-skeletal fluorosis group (control group). To collect venous blood 5 ml, whole blood DNA was extracted, and polymorphism at Rs1801282 of PPARγ was detected by MassARRAY time-of-flight mass spectrometry, to calculate odds ratio (OR) and 95% confidence interval (CI). Results There were 1 414 people included in this study,including 347 in case group and 1 067 in control group. By the Hardy-Weinberg balance test, the PPARγ gene Rs1801282 genotype was representative in case group, control group and each nationality (P > 0.05). The difference of PPARγ gene Rs1801282 genotype in case group and control group was not statistically significant (OR was 0.991, 95%CI: 0.704 - 1.395, the adjusted OR was 1.026, 95%CI: 0.707-1.489).The difference of PPARγ gene Rs1801282 genotype(CC,CG+GG)in case group and control group in different nationality was not statistically significant (Tibetan: OR was 1.400, 95%CI: 0.576 - 3.404, the adjusted OR was 1.258, 95%CI: 0.474 - 3.340; Kazak: OR was 0.898, 95%CI:0.516 -1.562,the adjusted OR was 0.936,95%CI:0.532 -1.648;Mongolia: OR was 1.148,95%CI:0.508-2.594, the adjusted OR was 1.644, 95%CI: 0.683 - 3.956; Han: OR was 1.058, 95%CI: 0.451 - 2.482, the adjusted OR was 0.959, 95%CI: 0.388 - 2.371; Russian: OR was 0.000, 95%CI: 0.000 - 0.000, the adjusted OR was 0.000, 95% CI: 0.000 - 0.000) with binary Logistic regression analysis. Conclusion We have found no association between SNP of PPARγ gene Rs1801282 and skeletal fluorosis of brick-tea type fluorosis in China.

Objective To study the urinary arsenic safety guideline value of a population for evaluating the arsenic exposure level in a certain population and providing evidence for the implementation of prevention and control measures in endemic arsenicosis area.Methods According to the data from the national high-arsenic drinking water sources screening in endemic arsenicosis area of drinking water type and quality supervision and inspection for water-improving project to decrease arsenic from 2005 to 2014,census data on arsenic poisoning in endemic arsenicosis area,data on surveillance of endemic arsenicosis,10 722 people with detailed personal information,complete water arsenic exposure data and accurate urinary arsenic detection data were selected to be the research objects.The relationship between urinary arsenic and water arsenic was analyzed based on the surveillance data of 4 501 people from 2013 to 2014.The safety guidance value of urinary arsenic was determined based on the geometric mean value of urinary arsenic in people exposed to water arsenic in the range of (0.050 ± 0.005) mg/L,and verified using the data of 6 221 people from 2005 to 2012.Every time,a random sample of 2 000 people was taken as the verification sample,the sensitivity and specificity of the index for determining whether water arsenic exposure exceeded the standard were determined by area under the ROC curve (AUC),and a total of 10 sample tests was performed.Results When the water arsenic concentration was less than 0.01 mg/L,the correlation coefficient of water arsenic concentration with urinary arsenic concentration was 0.097 (P ＜ 0.01);when the water arsenic concentration was more than 0.01 mg/L and less than 0.05 mg/L,the correlation coefficient of arsenic concentration with water arsenic concentration was 0.456 (P ＜ 0.01);when the water arsenic concentration was more than 0.05 mg/L,the correlation coefficient of water arsenic concentration with urinary arsenic concentration was 0.630 (P ＜ 0.01).With increase of water arsenic concentration,the concentration of urinary arsenic increased significantly,and the difference was statistically significant (x2 =2 337.956,P ＜ 0.01).When water arsenic concentration was in the range of (0.050 ± 0.005) mg/L,the urinary arsenic geometric mean was 0.032 mg/L.AUC analysis of 10 random samples of 2 000 people showed that the geometric mean of urinary arsenic was 0.032 mg/L in the population,which can accurately distinguish whether the water arsenic level exceeded 0.05 mg/L,and the AUC value was higher than 0.94.And the sensitivity and specificity were achieved 0.898 and 0.844.Conclusions The geometric mean of urinary arsenic is 0.032 mg/L,which can be used as a safety guideline value for urinary arsenic in the population.When the geometric mean of urinary arsenic exceeds this value,the population may be exposed to high arsenic.

Objective To observe the effect of arsenic exposure to drinking water on thelevel of histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 79 trimethylation (H3K79me3) in peripheral blood leukocytes of human,and to analyze the relationship between arsenic exposure and H3K4me3,H3K79me3 modification levels.Methods A cluster sampling survey was carried out in typical endemic arsenicosis areas of Shanxi and Jilin provinces.Two hundred eighty-one local residents with a drinking water age of ≥ 10 years were selected as the survey subjects.According to the arsenic content of drinking water,the tested population was divided into control group (water arsenic content ≤0.01 mg/L,60 cases),low water arsenic exposure group (＞ 0.01-0.05 mg/L,61 cases),medium water arsenic exposure group (＞ 0.05-0.10 mg/L,50 cases),and 110 cases of high water arsenic exposure group (＞ 0.10 mg/L).Drinking water samples,immediate urine samples and peripheral blood samples were collected from the subjects.Arsenic content in drinking water and urinary arsenic content were determined via the atomic fluorescence method;histone H3K4me3 and H3K79me3 in peripheral blood leukocytes were determined by dot blot hybridization (Dot Blotting).Results There were no statistically significant differences in age (61.50,60.00,59.50,59.50 years old),different gender (male:20,27,17,40 cases,female:40,34,33,70 cases),body mass index (BMI),smoking and drinking status between the control group,low,medium and high water arsenic exposure groups.Water arsenic content in the control group,low,medium and high water arsenic exposure groups (median:0.005,0.024,0.076,0.150 mg/L),urinary arsenic content (0.011,0.018,0.061,0.134 mg/L),and water arsenic cumulative exposure levels (0.342,1.641,5.273,7.716 mg) were compared between groups,the differences were statistically significant (H =256.041,88.615,218.610,P ＜ 0.01).In the control group,low,medium and high water arsenic exposure groups,the modification levels of H3K4me3 (0.100,0.059,0.083,0.083)and H3K79me3 (0.049,0.036,0.055,0.052) in peripheral blood leukocytes were not significantly different (H =1.488,2.097,P ＞ 0.05).The levels of H3K4me3 and H3K79me3 in peripheral blood leukocytes were positively correlated with water arsenic content,urinary arsenic content,water arsenic cumulative exposure levels (r =0.245,0.221;0.299,0.318;0.149,0.149;P ＜ 0.01 or ＜ 0.05);there was a positive correlation between H3K4me3 and H3K79me3 modification levels (r =0.811,P ＜ 0.01).Conclusion There is a positive correlation between arsenic exposure through drinking water and the levels of H3K4me3 and H3K79me3 in the peripheral blood leukocytes of the population,but it is necessary to expand the sample size for further study.

Objective:To explore the safety and efficacy of chimeric antigen receptor T-cell (CAR-T) therapy for relapsed/refractory acute B-cell lymphoblastic leukemia (B-ALL) with T315I mutation.Methods:The clinical data of a patient with relapsed/refractory B-ALL with T315I mutation who underwent CAR-T therapy in the Second Affiliated Hospital of Anhui Medical University was analyzed, and the related literature was reviewed.Results:The patient was a 34-year-old man. He was diagnosed with chronic myelogenous leukemia (CML) in January 2017 and started to take imatinib orally. However, the primary affection transformed to B-ALL 4 months later. Because of the E355G gene mutation, the treatment drug was adjusted to dasatinib, and induction chemotherapy was given at the same time. The sequential consolidation chemotherapy was given for 3 times after complete remission (CR). After half a year of remission, T315I mutation was detected and re-induced chemotherapy was given, but ineffective. The patient was treated with CAR-T 3 days after FC regimen (fludarabine 30 mg/m 2 per day, day 1 to day 3; cyclophosphamide 200 mg/m 2, day 1 to day 3). The number of CD19 CAR-T was 1.0×10 9, 98% activity degree. Grade 1 cytokine-releasing syndrome appeared after infusion, and was resolved after symptomatic treatment. No serious adverse reactions were observed. CR was achieved half-month after CAR-T treatment, and umbilical cord blood transplantation was successfully performed 1 month later. At the last follow-up, the relapse-free survival time of the patient was 396 days. Conclusion:CAR-T therapy may be a new, safe and effective therapy for patients with relapsed/refractory B-ALL with T315I mutation.

【Objective】 To identify and propose blood transfusion suggestions for 3 children suspected to have red blood cell T polyagglutination. 【Methods】 According to the RBC reactions with phytohemagglutinin, adult serum and cord blood serum, aggregation test with polybrene reagent and MN antigen phenotype test were carried out on 3 children to confirm the presence of T polyagglutination. The donor serum with negative or weak reactions was selected by minor cross matching for the 3 children who needed therapeutic plasma exchange(TPE). 【Results】 Three cases of RBC T polyagglutination were caused by bacterial infection, with transient appearance of MN antigen; the samples were reactive to peanut agglutinin, soybean agglutinin, adult serum but nonreactive to cord blood serum, and didn′t aggregate after adding polybrene reagent. After receiving timely TPE, the T polyagglutination gradually disappeared. 【Conclusion】 Some bacteria, such as Streptococcus pneumoniae, may cause polyagglutination of red blood cells. The patients with suspected T polyagglutination should be diagnosed in time. For T polyagglutination patients, the minor matched plasma should be used for avoiding the random plasma with anti-T antibody transfusion.

Objective To study the effect of fluoride exposure on bone growth in zebrafish.Methods The zebrafish larvaes at 3 days post fertilization (3 dpf) were exposed to the conventional fish water and 25,50,100 mg/L of NaF for 5 days until the skeletal bone was formed (8 dpf) and the temperature was kept at 28 ℃.The fluoride content of zebrafish embryos was detected by F-ion selective electrode.The fluoride exposure model was re-established as the control group (0.0 mg/L),the low doses group (0.5,1.0,4.0 mg/L) and the high doses group (50.0,100.0 mg/L).The survival rates of the zebrafish embryos were calculated and the morphology of zebrafish embryos was observed under 40 times microscope.The zebrafish skeleton was stained with alizarin red.The staining areas and the integrated optical density (IOD) of the bone staining were quantitatively analyzed by digital microscope to analyze the sclerotic and osteoporosis of the skull.Results The fluoride contents of the control group and 25,50,100 mg/L NaF groups were (0.32 ± 0.01),(0.63 ± 0.01),(0.86 ± 0.02) and (1.21 ± 0.01) μg/150 embryos.Compared with the control group,the fluoride contents of zebrafish embryos in fluoride exposed groups were increased (P ＜ 0.05),and the dose-response relationship was obvious.The survival rates of zebrafish embryos in control group and fluoride exposed groups were 96.67％,96.67％,96.67％,98.33％,98.33％ and 98.33％.There was no significant difference among different groups (x2 =7.309,P ＞ 0.05);under a 40 times microscope,there were no obvious deformities of the spin in different groups;the areas of the alizarin red staining of the skull were 84 380.51 ± 11 711.41 in the control group,92 592.16 ± 7 143.81,92 164.85 ± 10 136.18 and 95 112.26 ± 13 721.91 in the low doses exposure groups (0.5,1.0,4.0 mg/L NaF),67 778.92 ± 8 597.11 and 64 272.93 ± 9 302.57 in the high doses groups (50.0,100.0 mg/L NaF).The areas of the alizarin red staining of the skull in the low doses exposure groups were significantly higher than that of the control group (P ＜ 0.05),while the high doses exposure groups were lower (P ＜ 0.05);the IOD of the alizarin red staining of the skull was 25 094.13 ± 6 571.86 in the control group,29 754.95 ± 3 836.45,28 747.36 ± 4 677.86 and 30 776.49 ± 5 589.63 in the low doses exposure groups (0.5,1.0,4.0 mg/L NaF),19 263.10 ± 4 754.72 and 18 202.58 ± 4 897.15 in the high doses groups (50.0,100.0 mg/L NaF).The IOD of the alizarin red staining of the skull in the low doses exposure groups was significandy higher than that of the control group (P ＜ 0.05),while the high doses exposure groups was lower (P ＜ 0.05).Conclusion Low doses of fluoride exposure may cause bone sclerosis in zebrafish embryos,while the high dose of fluoride exposure may cause osteoporosis.

Objective To observe the effect of fluoride on fibroblast growth factor 23 (FGF23) in bone tissue of mice,and to explore the role of FGF23 in fluoride-induced bone injury.Methods Sixty-four Balb/c mice,half male and female,were divided into 4 groups based on body weight via the random number table method and 16 mice were in each group.The mice in control group,low fluoride group,middle fluoride group and high fluoride group were treated with 0,25,50,and 100 mg/L F-distilled water,respectively.After three months,the mice were put to death and the prevalence of dental fluorosis was calculated.The fluoride contents in spine were detected via the fluoride-ion selective electrode method,serum content of calcium and phosphorus were detected by micro enzyme labeled method.The levels of FGF23,parathyroid hormone (PTH) and 1,25 dihydroxy vitamin D3 [1,25 (OH)2D3] in serum were measured by enzyme-linked immunosorbent assay.The FGF23 protein expression levels in bone tissue were determined by immunohistochemistry and Western blotting.Results The rates of dental fluorosis in low,medium and high fluoride groups were 75％ (4/16),100％ (16/16) and 100％ (16/16),respectively.Compared with control group [0 (0/16)] the differences were statistically significant (P ＜ 0.05).The levels of fluoride in the fluoride group [low,medium,high fluoride groups:(1 730.86 ± 165.90),(2 400.58 ± 286.65),(3 980.88 ± 511.65) mg/kg] were higher than that of control group [(854.30 ± 89.05) mg/kg,P ＜ 0.05].There was no difference in serum calcium content among groups (F =0.05,P ＞ 0.05).The contents of phosphorus in the serum of the medium and the high fluoride groups [(2.46 ± 0.32),(2.48 ± 0.73) mmol/L] were lower than those in the control and the low fluoride groups [(2.89 ± 0.45),(3.25 ± 0.69) mmol/L,P ＜ 0.05].The serum PTH and 1,25 (OH)2D3 content increased first and then decreased.The expression of FGF23 in middle and high fluoride groups [(660.84 ± 64.18),(638.74 ± 121.23) ng/L] was up-regulated compared with that of control group [(613.53 ± 98.18) ng/L].The expression of FGF23 protein in cortical bone increased gradually with the dose of fluoride.Western blotting results showed that the content of FGF23 protein in the bone tissue of mice was significantly increased in the low fluoride group (1.58 ± 0.46) and the middle fluoride group (1.40 ± 0.41) compared with that of control group (1.00 ± 0.41),the differences were statistically significant (P ＜ 0.05).Conclusions The phosphorus,FGF23,PTH,and 1,25 (OH)2D3 levels in the serum and FGF23 protein levels in the bone tissue of fluorosis mice have changed.It may be suggested that FGF23 interacts with PTH and 1,25 (OH)2D3 to influence the level of calcium and phosphorus metabolism in the body and participate in the formation of skeletal fluorosis.