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Objective:To explore the DNA methylation patterns and methylation differential genes of patients with coal-burning-borne endemic fluorosis, and to provide a basis for study of the pathogenesis of fluoride-induced body injury.Methods:A case-control study was conducted in Shuicheng County, Liupanshui, Guizhou Province, ten patients with severe fluorosis were selected as the fluorosis group in Douqing Township, where people burning high fluorine coal in open range all year round; and ten people without fluorosis phenotype were selected as the control group in Huaga Township, where firewood was the main fuel. Peripheral blood samples were collected from the two groups of people. Reduced representation bisulfite sequencing (RRBS) technique was used to detect the whole genome DNA methylation pattern ( n = 4) and DNA differentially methylated region (DMR), the DMR differential degree (log 2Ratio) and KEGG pathway enrichment analysis were used to screen the methylation differential genes, and real-time PCR was used to verify the mRNA expression levels of the candidate methylation differential genes( n = 10). Results:The methylation pattern analysis results showed that the methylation levels of all C bases in the genome DNA of the fluorosis group and the control group were (61.53 ± 0.59)% and (62.48 ± 1.53)%, respectively; among them, the methylated levels at CG sites were (63.75 ± 0.65)%, (64.36 ± 1.01)%, at CHG sites were (13.79 ± 0.72)%, (16.69 ± 4.06)%, and at CHH sites were (25.12 ± 1.72)%, (29.77 ± 3.97)%. Compared with the control group, patients in the fluorosis group had 1 000 DMR distributed on different autosomes; and the chromosome 19 was the most with 104 segments. There were 978 DMR-related genes, including 265 hypermethylation genes and 713 hypomethylation genes; KEGG pathway enrichment analysis showed that methylation differential genes were mainly involved in cell metabolism, cancers, and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and other signaling pathways; combined with the differential degree of DMR, the hypermethylated succinate dehydrogenase complex flavoprotein subunit A pseudogene 3 (SDHAP3, log 2Ratio = 3.487) and hypomethylated nuclear factor κB inhibitor kinase regulatory subunit γ (IKBKG, log 2Ratio =-4.436) were selected as the candidate genes. There were statistically significant differences in the mRNA expression levels of SDHAP3 (0.54 ± 0.08, 1.00 ± 0.00) and IKBKG (1.32 ± 0.39, 1.00 ± 0.00) between fluorosis group and control group ( F = 22.94, 15.09, P < 0.01 or < 0.05). Conclusion:There are a large number of methylation differential genes in the genomes of patients with coal-burning-borne endemic fluorosis and controls, the hypermethylated SDHAP3 and hypomethylated IKBKG may be involved in fluoride induced body injury.
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Objective To investigate the bacterial vaginosis(BV) and mycoplasma infection status in female patients with infertility.Methods The vaginal discharge samples were collected from 541 female patients with infertility in the gynecological department of the Hainan Provincial People's Hospital from June to December 2013.The BV detection,mycoplasma culture and drug susceptibility test were simultaneously conducted.Results The mycoplasma culture positive rate was 44.5％ (241/541),in which ureaplasma mycoplasma (UU) had the highest detection rate,there were 223 cases ofinfection with the positive rate of 41.2 ％,there were 5 cases of mycoplasma hominis(Mh) infection with the positive rate of 0.9 ％,13 cases were UU + Mh mixed positive with the positive rate of 2.4％.BV infection was in 325 cases with the positive rate of 60.1％ (325/541).Among 241 cases of mycoplasma positive,the BV detection rate was 70.1％ (169/241),while among 300 cases of mycoplasma negative,the BV detection rate was 52.0％ (156/300),the BV detection rate had statistical difference between the mycoplasma positive group and mycoplasma negative group,the difference was statistically significant (P＜0.05).Ofloxacin in fluoroquinolones had the highest drug resistance,the sensitivity was only 7.1％.Josamycin had the lowest drug resistance,accounting for 3.7 ％.Conclusion The female patients with infertility is closely correlated with BV and mycoplasma infection.Therefore BV and mycoplasma census should be strengthened to achieve early diagnosis and early treatment.Mycoplasma drug susceptibility test shows that doxycycline,minocycline and josamycin have the highest sensitivity,which can be used as the first selection drugs for clinical doctor.
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Objective:To prepare nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR and to examine its cytotoxicity against EGFR positive tumor cells.Methods:By using molecular cloning strategy,prokaryotic expression construct of pET28a-BI7D12-PE38KDEL was generated which consisted of nanobody 7D12 targeting EGFR in the form of a divalent fused with PE38KDEL,a truncated form of pseudomonas exotoxin A via a flexible peptide(G4S)4,and then transformed into E.coli BL21(DE3).Protein expression was induced by adding IPTG,purified by Ni-affinity column chromatography,and verified by Western blot.The binding capacity of the resulted immunotoxin to EGFR-positive cells A549,HT29,MCF-7 and EGFR-negative cells CEM,Jurkat were determined by flow cytometry assay,and its cytotoxicity against the target cells was examined.Briefly,tumor cells were treated with different dosage of the immunotoxin,and the killing efficacy of BI7D12-PE38KDEL on these cells were assessed by WST-1 assay after 72 hours.Results:The SDS-PAGE and Western blot results showed the recombinant immunotoxin BI7D12-PE38KDEL was successfully prepared,and majority of them was expressed in soluble form.BI7D12-PE38KDEL could selectively bind to EGFR-positive cells of A549,HT29,and MCF-7.More importantly,the immunotoxin exhibited much more significant killing effect on these EGFR positive cells compared to the negative control group of CEM and Jurkat cells(P<0.01).Conclusion:In the current study,the nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR was successfully prepared and exhibited a superior inhibition effect for the growth of EGFR-positive cells.
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Objective To observe the latency and amplitude characteristics of event-related potential P300 under different target stimu-lus. Methods During January to December, 2014, 35 patients with memory impairment received P300 test under the percentage of target stimuli of 0.10 and 0.25. The latency, amplitude and area under amplitude were compared. Results There was no significant difference in la-tency (t0.800), but the amplitude and area under amplitude increased under 0.10 compared with those under 0.25 (t>4.259, P<0.05). Conclusion Less target stimulation may improve the patients' attention and increase amplitude of P300, but no more interference in la-tency, suggesting latency of P300 is more stable for assessment of cognitive function.