Objective:To Clone the silencer sequences of human stimulator of interferon genes (STING) and evaluate its activity in HEK293T and HeLa, and to preliminarily investigate the transcriptional regulatory mechanisms, screening and verifying the possible binding elements for the silencer sequences of STING.Methods:The human STING-5-1a(-124~+ 267, transcription start site, TSS: 0) and STING-5-2a(-124~+ 168) regions were amplified by PCR, subcloned into pGL3-Basic plasmid, and the luciferase activity was detected in HEK293T and HeLa; the human STING silencer region STING-5-1b(+ 169~+ 267) was subcloned into the pGL3-Control plasmid (pGL3-C-5-1b-positive/negative) and STING-5-1b is divided into two complementary elements, subcloned into pGL3-Control vector, named pGL3-C-STING 5-1b-α(+ 169~+ 209) and pGL3-C-STING-5-1b-β(+ 210~+ 267), detecting the relative luciferase activity of the above plasmid in HEK293T and HeLa. Using bioinformatics methods to predict the transcription factor binding site of human STING silencer, make site-directed mutagenesis at the predicted binding site, and detect luciferase activity. Knockdown of THAP1 and TEAD1 by a siRNA strategy and detect the expression level of STING by western blot analysis. Chromosome immunoprecipitation assay further verified the binding of transcription factor to hSTING silencer.Results:It was verified that plasmids mentioned above were constructed correctly by nucleotide sequencing. The relative luciferase increased after truncating the STING-5-1b(+ 169~+ 267) fragment. The relative luciferase activity of pGL3-C-5-1b-positive recombinant plasmid decreased ( P<0.05), compared with pGL3-Control plasmid. Among them, STING-5-1b-β(+ 210~+ 267) fragment play a major inhibitory role. Using bioinformatics software to predict that the transcription factors PR domain zinc finger protein 4 (PRDM4), Thanatos-associated protein 1 (THAP1), TEA Domain Transcription Factor 1 (TEAD1), Nuclear receptor subfamily 4 group A member 1 (NR4A1), Krueppel-like factor 4 (KLF4) and Forkhead box protein O3(FOXO3) may bind to the human STING silencer region (+ 210~+ 267). After transfecting the mutant recombinant plasmid of the transcription factors into HEK293T and HeLa, the relative luciferase activity of THAP1-Mut and TEAD1-Mut were significantly increased, suggesting that STING silencers may contain binding sites of THAP1 and TEAD1. Knockdown of THAP1 and TEAD1 by a siRNA strategy significantly enhanced the transcription activity. Chromosome immunoprecipitation assay showed that the transcription factors TEAD1 and THAP1 combined with hSTING silencer region in the cells. Conclusions:The hSTING silencer luciferase reporter plasmid was successfully constructed. By the activity comparison, it is speculated that the core silencer region of human STING is located in the + 210~+ 267 element, which may contain several potential transcription factor binding sequences.The potential binding sites for transcription factors that may be contained in the DNA, and use Western blot and chromosome immunoprecipitation assays to further confirm the combination of transcription factors TEAD1 and THAP1 with hSTING silencer, laying the foundation for subsequent research.

Objective To identify the animal drug of Cervus elaphs and C. nippon from origin of deers. Methods To extract DNA from deer blood and hairy antler of 11 species of deers such as C. elaphus, C. nippon and so on, and to gain the mitochondrial 12S rRNA gene fragment using the general primers of L1091 and H1478. Based on the sequence multialinement of 11 species deers above gene fragments, designing the couples of special difference primers and identifying C. elaphs and C. nippon. Results 12S rRNA Gene fragments can distinguish different deers well. The couple of primers (EP-1/H1478 and EP-2/H1478) PCR can effectively identify C. elaphus and C. nippon. Conclusion Special primer PCR is suitable for the identification of valuable Chinese medicinal materials, such as C. elaphus and C. nippon.

Objective:To analyze the application effect of three-fourths prone position drainage method in patients with pulmonary infection and consciousness disorders after tracheotomy.Methods:A total of 84 patients with consciousness disorders who were admitted to the First Affiliated Hospital of Wenzhou Medical University from January 2018 to October 2020 with pulmonary infection after tracheotomy were selected. They were divided into the control group and the observation group, there were 42 cases in each group according to random number table method. The control group received routine prone position drainage for pulmonary infection after tracheotomy and the observation group was given three-fourths prone position drainage method. The arterial partial pressure of oxygen(PaO 2), arterial partial pressure of carbon dioxide(PaCO 2) before and after intervention, the drainage effect after the intervention, the absorption of pulmonary infection foci, and the time of antibiotic treatment for pulmonary infection during the patients′ hospitalization were compared between the two groups. Results:After the intervention, PaO 2 and PaCO 2 were (91.87 ± 7.21), (35.34 ± 3.28) mmHg(1 mmHg=0.133 kPa) in the observation group, and (85.23 ± 7.90), (43.41 ± 3.39) mmHg in the control group, the differences between the two groups were statistically significant ( t=-4.02, 11.09, both P<0.05). After the intervention, the apparent rate, effective rate, and ineffective rate were 78.57%(33/42), 19.05% (8/42), 2.38% (1/42) in the observation group,and 33.33% (14/42), 45.24% (19/42), 21.43% (9/42) in the control group. The drainage effect of the observation group was better than that of the control group, and the difference was statistically significant ( Z=-4.28, P<0.05). After the intervention, the complete absorption rate of the pulmonary infection foci and the time taken to treat pulmonary infection with antibiotics during hospitalization were 59.52% (25/42), (10.67 ± 2.70) d in the observation group, and 35.71%(15/42), (13.51 ± 3.46) d in the control group, the differences were statistically significant ( χ2=4.77, t=4.19, both P<0.05). Conclusions:The three-fourths prone position drainage method has significant application effect in patients with pulmonary infection and consciousness disorder after tracheotomy. It can effectively improve the drainage effect, improve oxygenation, promote the absorption of lung infections, and shorten the antibiotic treatment time.

Objective:To assess the prognostic value of methylation of interferon regulatory factor 6 ( IRF6) gene promoter in patients diagnosed with Kidney renal clear cell carcinoma (KIRC). Methods:The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected. The expression of IRF6 protein was determined with an immunohistochemical method. The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed. The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools. The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases. Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival (OS) among the patients. In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene. Results:The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls, and this was more prominent in patients who had died or developed metastasis. The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues. There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6 ( R= -0.52). The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177, the shorter the OS and worse prognosis in the patients. Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines. The transcription level of the IRF6 gene was upregulated in a time- and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine. Conclusion:The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS. Cg12034118 may provide a promising biomarker for laboratory detection, and its high methylation rate has certain reference value for the prognosis.

OBJECTIVETo assess the detection of maternal serum alpha fetoprotein (MSAFP) and free beta-HCG levels of second trimester for screening of fetal gastroschisis and omphalocele.

METHODSClinical data of 622 639 pregnant women from 5 prenatal screening centers in Hangzhou during October 2007 and September 2016 were analyzed retrospectively. Thirty cases of gastroschisis and 30 cases of omphalocele diagnosed by ultrasonography and postmortem findings were enrolled in the study and 116 cases of pregnant women with normal fetal development during the same period were selected as control group. The cut-off value and area under ROC curve (AUC) of MSAFP and free β-hCG for diagnosis of fetal gastroschisis and omphalocel were analyzed.

RESULTSMSAFP levels of women with fetal gastroschisis and omphalocele were 4.41 (0.88-11.69) MOM and 2.31 (0.72-23.20) MOM, which were significantly higher than that of control group[0.98 (0.41-2.26) MOM, all<0.01]. Free β-hCG level of women with fetal gastroschisis was 1.25 (0.35-19.94) MOM, which was significantly higher than that of control group[0.86 (0.17-6.11) MOM,<0.05). But there were no significant difference in free β-hCG between fetal omphalocele group[1.03(0.21-8.95)]and control group (>0.05). The AUCs of MSAFP for diagnosis of gastroschisis and omphalocele were 0.897 (95%:0.822-0.972) and 0.852(95%:0.762-0.942), respectively (all<0.01). Taking 1.655 MOM as the cut-off value of MSAFP for abdominal wall defects (gastroschisis and omphalocele), the sensitivity was 68.30%, specificity was 99.60% and Youden index was 0.649.

CONCLUSIONSMSAFP of second trimester is a better biomarker than free β-hCG in screening abdominal wall defects.

OBJECTIVE: To explore the characteristics of mutations of four common pathogenic genes (GJB2, SLC26A4, GJB3 and 12S rRNA) among patients with nonsyndromic hearing loss (NSHL) from eastern Shandong. METHODS: Peripheral blood samples of 420 NSHL patients were collected, and a hereditary-deafness-gene microarray was used to detect GJB2 c.235delC, c.299-300delAT, c.35delG and c.176del16 mutations, GJB3 c.538C>T mutation, SLC26A4 c.2168A>G and c.IVS7-2A>G mutations, and 12S rRNA c.1555A>C and c.1494C>T mutations. For patients carrying single heterozygous mutations, the coding regions of the above genes were analyzed with Sanger sequencing. RESULTS: The results of the microarray assay and Sanger sequencing showed that 84 patients (20.00%) carried GJB2 mutations, with c.235delC (16.43%) and c.299-300delAT (7.86%) being most common. Seventy-five patients (17.86%) carried SLC26A4 mutations, for which c.IVS7-2A>G accounted for 15.71%. In addition, 5.95% of patients carried 12S rRNA mutations. Only one patient was found to carried GJB3 mutation (c.538C>T). CONCLUSION Common pathogenic mutations for NSHL in eastern Shandong included GJB2 c.235delC and SLC26A4 c.IVS7-2A>G. Of note, 5.95% of patients were due to 12S rRNA m.1555A>G mutation, which gave a frequency greater than other regions of China.
China ; Connexin 26 ; Connexins ; DNA Mutational Analysis ; DNA, Mitochondrial ; Deafness ; Genes, rRNA ; Hearing Loss ; Humans ; Mutation ; RNA, Ribosomal ; Sulfate Transporters