Identification and characterization of stimulator of interferon gene silencer / 中华微生物学和免疫学杂志

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.

Establishment of Reference Interval for Serum Prostate-Specific Antigen (PSA) of Apparent Healthy Men in Nanjing / 现代检验医学杂志

Objective To establish the reference interval for serum prostate-specific antigen (PSA) in apparent healthy men of different ages in Nanjing.Methods A total of 25 820 healthy men undergoing routine physical examinations in the First Affiliated Hospital of Nanjing Medical University from October 2013 to September 2015 were selected for the study.All of them were screened by prostate B ultrasound,excluding abnormal urinary tract diseases.The concentration of serum PSA and free prostate-specific antigen (fPSA) were measured by automatic luminescence immunoassay analyzer,and the fPSA/PSA values were calculated.The participants were divided into four groups (20～ 39,40～ 59,60～ 79 and older than 80 years old groups),then the median,5th,25th,75th and 95th percentiles of both PSA and fPSA/PSA were counted,respectively.Results The median of PSA (95th percentile ranges) of these groups by age from low to high were 0.78 (1.93),0.90 (2.93),1.34(6.60) and 2.01(11.91),respectively.The 25th to 75th percentiles were 0.55～1.11,0.61～1.36,0.77～2.51 and 0.94 ～ 4.19,respectively.The median of fPSA/PSA (95 th percentile ranges) were 0.37 (0.60),0.31 (0.56),0.28 (0.53) and 0.29(0.52),respectively.The 25th to 75th percentiles were 0.28～0.46,0.23～0.40,0.22～0.36 and 0.23～ 0.37,respectively.Among all the groups,median differences of both PSA and fPSA/PSA were statistically significant (P＜0.05),and PSA levels rise with age.PSA levels in different regions were different.Conclusion The PSA level of men under 40 years in Nanjing should be 0～2.5 ng/ml,40～60 years should be 0～4 ng/ml,while men who are above 60 years,could use 0～5 ng/ml as reference interval.

Comparative Diagnostic Value of Chitinase 3-Like 1 Protein and AFP in the Diagnosis of Hepatocellular Carcinoma / 现代检验医学杂志

Objective To investigate the different diagnostic value of serum Chitinase 3-like 1 protein(CHI3L1)and Alpha-fe-toprotein(AFP)in diagnosing hepatocellular carcinoma (HCC).Methods One hundreds HCC patients confirmed by histopa-thology were recruited between December,2015 to April,2016 from the First Affiliated Hospital of Nanjing Medical Univer-sity.Simultaneously,100 patients with chronic hepatitis B and 59 healthy individuals,matched by sex and age with HCC pa-tients,were recruited as control groups.Serum CHI3L1and AFP were measured in different groups and the difference were analyzed by STATA 1 2.0 Statistical software.The ability of these two items in differentiating different group was analyzing by ROC curve using MedCal Ver 15.2.2 software.Results Serum CHI3L1 were significantly differences in the three groups using Kruskal-Wallis analysis (χ2=93.19,P=0.000),the differences were further compared in different two groups using Mann-Whitney analysis.The results showed that serum CHI3L1 in HCC group were significantly higher than in chronic hepatitis B and healthy control group (P=0.000,z=8.766,7.400).Serum AFP were significantly differences in the three groups using Kruskal-Wallis analysis (χ2=147.54,P=0.000),and the differences were further compared in different two groups using Mann-Whitney analysis.The results showed that serum AFP in HCC group were significantly higher than in chronic hepatitis B and healthy control group (P=0.000,z=10.938,9.033).The ROC curve analysis of serum CHI3L1 and AFP for differentiating HCC group from CHB group showed that CHI3L1 yield AUC of 0.859 (95% CI:0.803~0.904) with 85% sensitivity,79% specificity and 76.8 pg/ml cut-off value,AFP yield AUC of 0.948 (95% CI:0.904~0.974)with 85% sensitivity,98% specificity and 7.6 ng/ml cut-off value,in distinguishing HCC with CHB group,the power of AFP was superior to that of CHI3L1 (P=0.006).The ROC curve analysis of serum CHI3L1 and AFP for differentiating HCC group from healthy individuals group showed that CHI3L1 yield AUC of 0.852 (95% CI:0.787~0.903)with 85% sensi-tivity,76% specificity and 76.8 pg/ml cut-off value,AFP yield AUC of 0.929 (95% CI:0.878~0.964)with 84% sensitivi-ty,100% specificity and 7.8 ng/ml cut-off value,in distinguishing HCC with healthy individuals group,the power of AFP was also superior to that of CHI3L1 (P=0.045).Conclusion Serum CHI3L1 similar to AFP has much power ability to di-agnosis HCC,but AFP was superior to CHI3L1.

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA

BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

Structure and Function of an Alternative Splicing Isoform of Stimulator of Interferon Genes STING(sv) / 病毒学报

Stimulator of interferon genes (STING) is an important protein of the innate immune response, and protects against viral infections. To search for an alternative splicing isoform of STING, we undertook rapid amplification of cDNA ends (RACE) and RT-PCR with RNA extracted from human embryonic kidney (HEK) 293 cells and primers designed according to the mRNA sequence of full-length STING(NM-198282. 82). The new sequence was compared using a bioinformatics method. Then, a newly discovered, alternative splicing isoform of STING, named "STING(sv)", and STING(wt) were subcloned into the eukaryotic expression vector pEGFP-C1 and pcDNA 3. 1. Whole-cell extracts were analyzed by western blotting and then probed with monoclonal antibody against enhanced green fluorescent protein (EGFP) after transfection of EGFP-STING(wt) and EGFP-STING(wt) plasmids in HEK293 cells. pcDNA-STING(wt) and pcDNA-STING(wt) were transfected in HEK293 cells, and the luciferase assay carried out. Compared with STING(wt), STING(sv) lacks exon 7 so that shift in the reading frame may produce a protein with a different C-terminal in amino acids 1-30. Western blotting confirmed an expected strong band at 58 x 10(3) kD. The functional luciferase assay showed that STING(sv) inhibited the activity of the interferon (IFN)-β promoter. STING(sv) can be expressed in multiple tissues and distinct cell lines. Our discovery of a new, alternative splicing isoform of STING provides new insights into the functional regulation of STING. STING(sv) could be a dominant negative inhibitor for the activity of the IFN-β promoter in the virus-infection pathway. Hence, STING(sv) could participate in the "fine tuning" of the virus-induced activation of IFN. Therefore, exploring the role of STING(sv) in the pathogenesis of human diseases could be very worthwhile.

Results analysis of blood infectious indicators in 331 968 outpatients and inpatients / 国际检验医学杂志

Objective To analyze the results of detection of infectious indicators of patients from the first affiliated hospital of Nanjing Medical University in recent 5 years ,and to provide a scientific basis for the control and prevention of infectious diseases . Methods The patients with clinical data from January 2010 to April 2014 were retrospectively analyzed ,then the infectious indica‐tors of all the subjects were detected and analyzed .Results HIV positive rate was between 0 .043% to 0 .061% ,positive rate of HCV was between 1 .07 to 1 .41% ,positive rate of TP was between 2 .01% to 2 .17% .HBsAg positive rate in 2010 was 8 .36% ,the positive rate was 7 .81% in 2014 .HBsAb positive rate in 2010 was 35 .36% ,positive rate was 50 .96% in 2014 .Conclusion Effec‐tively cut off the route of transmission could prevent the further spread of infectious disease .

Application of Bone Gamma-Carboxyglutamic-Acid-Containing Protein(BGP)in Middle-Aged and Elder People’s Health Care / 现代检验医学杂志

Objective To evaluate differences of serum bone gamma-carboxyglutamic-acid-containing protein (BGP)levels be-tween different gender in the middle-aged and elder population and the correlation between serum BGP and osteoporosis,as well as the correlation between BGP and other bone metabolic markers.Methods The study population consisted of 270 health care middle-aged and elder people,who were excluded malignancy and chronic diseases,during 2011 January to Au-gust.Of all the Recipients,101 cases were male,aged 50 to 89 years,with a median age of 68 years old,female 169 cases, aged 50 to 89 years,with a median age of 64 years.Bone density were measured by absorptiometry and was evaluated by the T index values.Serum BGP,25-hydroxyvitamin D,calcium and phosphorus were measured by different assays systems.The different level of BGP between genders was also analyzed by Mann-Whitney test.Correlation between BGP and serum calci-um,phosphorus,25-hydroxyvitamin D,osteoporosis risk index were analyzed Spearman rank correlation analysis.Results T value,BGP,25-hydroxy vitamin D,calcium and phosphorus levels range of 270 cases were-3.5~-0.7 (median-1.6 ng/ml),3.59~264.90 ng/ml (median 12.84 ng/ml),4.0~34.0 ng/ml (media 10.5 ng/ml),1.79~2.69 mmol/L (median 2.36 ng/ml),0.43~2.89 mmol/L (median 1.12 ng/ml),respectively.BGP levels in the female groups were significantly higher than the male groups.Serum concentration of BGP was positively correlated with serum phosphorus,but the serum BGP with calcium,25-hydroxy vitamin D,age and osteoporosis risk indices were not correlated.Conclusion In the elder groups,female BGP levels were significantly higher than male,the gender factor should be considered in the clinical applica-tion of BGP.Since BGP and osteoporosis risk index T had positive correlation,those two tests can be combined to evaluate osteoporosis.

Difference of serum 25-hydroxyl vitamin D concentration level in common autoimmune diseases / 国际检验医学杂志

Objective To investigate the serum concentration of 25-hydroxyl vitamin D in the common autoimmune diseases and whether the differences of the 25-hydroxyl vitamin D level exist in different autoimmune diseases.Methods 137 cases of autoim-mune diseases,including 71 cases of rheumatoid arthritis(RA),36 cases of systemic lupus erythematosus(SLE),16 cases of Sjogren syndrome(SS)and 14 cases of ankylosing spondylitis(AS),in our hospital from January 2012 to April 2013 were selected as the re-spondents.The serum samples were collected for detecting the 25-hydroxyl vitamin D level by the electrochemiluminescence method and comparing the differences of the 25-hydroxyl vitamin D level among different autoimmune diseases.At the same time whether the differences in the proportion of the normal level,insufficiency and lack of 25-hydroxyl vitamin D exist among different kinds of disease.Results The serum 25-hydroxyl vitamin D concentration had statistically significant difference among the patients with dif-ferent autoimmune diseases(P =0.006),which in the RA group was significantly higher than that in the other groups;the propor-tion of 25-hydroxyl vitamin D insufficiency in RA,SLE,SS and AS were 29.6%,52.8%,62.5% and 57.1% respectively,which in the SLE,SS and AS groups was significantly higher than that in the RA group(P <0.05).Conclusion The 25-hydroxyl vitamin)D insufficiency is general in common autoimmune diseases,the vitamin D supplements needs to be strengthened.

Construction of X IAP-3′UTR-luciferase reporter vector and its activity analysis / 国际检验医学杂志

Objective To construct the recombinant X-linked inhibitor of apoptosis protein(XIAP) gene 3′untranslational region (3′UTR)-luciferase reporter vector ,and analyze the microRNA(miRNA) which possibly regulate the expression of XIAP gene . Methods Polymerase chain reaction (PCR) was employed to amplify X IA P-3′UTR sequences from human cDNA ,in which luciferase reporter vector pGL3-Ctrl was inserted ,and the recombinant vector pGL3-Ctrl/XIAP was gained .Target Scan 6 .2 soft-ware was adopted to predict the miRNA which possibly combined with the X IA P-3′UTR .pGL3-Ctrl/XIAP recombinant plasmids and the miRNA were co-transfected into A549 cells ,and the X IA P-3′UTR-luciferase activity was measured .Results Confirmed by digestion and DNA sequencing ,the X IA P-3′UTR-luciferase reporter recombinant was successfully constructed .Prediction of miRNA target sites indicated that X IA P gene may be the target of miR-200b ,miR-200c and miR-429 .Compared with miRNA mim-ic ctrl group ,miR-200b ,miR-200c and miR-429 significantly reduced the luciferase activity of pGL 3-Ctrl/XIAP with statistically significant difference(P<0 .05) .Conclusion X IA P-3′UTR-luciferase reporter vector is successfully constructed .miR-200b ,miR-200c and miR-429 can obviously decrease the luciferase activity .

Characterization of promoter of human interferon regulatory factor 3 / 中华微生物学和免疫学杂志

ays revealed that the-(167-111)bp region Was the minimal promoter of the human IRF3 gene.These results suggested that transcriptional factors such ag E2F might be involved in the transcriptional regulation of IRF3 gene.