| Steroid hormone receptors(SHRs),which belong to the nuclear receptor superfamily,are ligand-dependent transcription regulators that can regulate the growth,development and metabolism of organisms by regulating gene expression.SHRs have played a main role in a wide range of pathophysiological conditions,such as prostate cancer,breast cancer and familial/sporadic glucocorticoid resistance.Vast numbers of disease-associated alternations have been identified,which occurred frequently in related disease prior to hormonal therapy but become prevalent in incurable hormone independent and metastatic tumors.The conventional dual reporter system assay which contains one experimental luciferase reporter gene and one control luciferase reporter gene in a single assay is a sensitive and fast option for identifying potential functional variants in vitro.Inevitable limitations of this approach including low transfection efficiency when three different vectors were need to be transfected simultaneously,uneven expression level of these three vectors and different outcomes that may induced by different ratios of these three vectors at different experimental conditions.Therefore,advances in conventional dual reporter system assay are urgently needed.We constructed recombinant plasmids which contain an effective promotor gene upstream the firefly luciferase reporter gene and SHR wide type or SHR variants or SHR mutations genes upstream of the Renilla luciferase reporter gene,and to perform the dual-luciferase reporter assays to study transcriptional abilities of various AR,ER? and GR alternations that constructed based on previous clinical reports.The results revealed that nine AR variants(AR-V4,AR-V5,AR-V6,AR-V7,AR-V8,AR-V9,AR-V11,AR-V12,and ARQ640S)and three AR mutations(W742C,T877A and W742C-T877A)were found to have constitutive activity in the absence of R1881;As for Er?,six ER mutations(S463P-D538G,Y537N-D538G,Y537S-D538G,Y537N,Y537S and D538G)and one Era variants(Er?-V5)exhibited strong constitutive transactivation;one mutations(Y537K and D538N)and four ER-Vs(TIDDA,Er?-36,Er?-46,Er?-V6)showed higher transactivation ability compared with the wide-type Era in the presence of estrogen but similar function in the absence of estrogen;among the various GR alternations,only GR-B displayed constitutive transactivation.This study showed that the new sensitive assay based on the conventional dual reporter system can be used to identify the functionality of variations and mutations.The new system uncovered a previously unidentified or identified category of SHR(AR,Era and GR)mutations and variants with distinctive functional properties at physiological levels.The recent discovery of recurrent SHR alternations in related disease may merely represent the tip of the iceberg but do provide the basis for further exploration of hormone-independent progression and endocrine therapy resistance mechanisms.Human T-lymphocytotropic virus(HTLV)was the first RNA retrovirus found to be associated with cancer.Adult T-cell leukemia(ATL)and HTLV-associated myelopathy/tropical spastic paraparesis(HAM/TSP)are the two most common pathologies developed from HTLV-1.It was clear that HTLV was mainly transmitted by mother-to-child,sexual contact and blood transfusion.Mandatory screening of blood supplies for HTLV-1/2 was implemented in most developed and several developing countries.Western Blot and line immunoassay are the most frequently used for HTLV-1/-2 confirmation for the initially reactive samples.Unfortunately,a large number of indeterminate and HTLV-positive but untypeable results are commonly identified,which are difficault to confirm the real status of the individuals.and this method cannot quantify the proviral load(PVL)for each detection sample.Nevertheless,quantify the proviral load(PVL)can not only confirm the real status of each detection sample,but can also understand clinical outcomes and evaluate therapeutic intervention of HTLV-1 infected patients.Real-time quantitative PCR(qPCR)technology that is based on TaqMan probe with high sensitivity and specificity is widely used for nucleic acid detection and quantification.In this part,multiple pairs of primers and probes were designed based on the HTLV-1 conserved region(pol region and tax region)and the internal reference RPPH1 gene.Then we optimized the concentrations and annealing temperature of the selected primers and probes to establish a method for quantitative detection of HTLV-1 provirus.In this study,the quantitative test results used the number of peripheral blood mononuclear(PBMC)cells as the denominator and the number of HTLV-1 infected cells as the numerator.Subsequently,we based on the copy number of the internal reference gene RPPH1 in each PMBC as 2 to relatively quantify the copy number of HTLV-1 of each cell.Fanilly,we performed a series of methodological evaluations on the established qPCR method and quantified 41 clinical samples by using this method.The result showed that the method owned 100%specificity and the detection scale of qPCR is ranging from 15 to 2.5×108copies per reaction,which is sensitive to be applied in clinic.The digital PCR showed the average copy number of HTLV-1 in Hut102 cells,which is a T cell lymphoma cell line that can release HTLV-1,is 1.146 copies/cell.Then the Hut102 cells were mixed with Jurkat,a T-cell lymphoma cell without HTLV-1,in different proportions.The limit of detection(LOD)by using the established qPCR method in this study for Hut102 cell concentration was 0.0218%(95%confidence interval 0.0179?0.0298%),which means the detection limit for the HTLV-1 pol region was estimated to be 2.5 copies per 10,000 host leukocytes with a 95%hit-rate.The qPCR method was used for quantifying 41 whole blood samples with LIA-indeterminate results(30 cases)and positive results(11 cases).The result showed that 7 of 11 LIA positive samples were HTLV-1 positive samples(including 1 LIA-untyped sample).The average PVL was 3.10 copies/100 cells;4 of the 7 samples were WB-HTLV-1 positive and 3 were uncertain.The results showed that the sensitivity of qPCR method is higher than WB,and could be used for typing and quantification of the HTLV positive samples.All of the LIA-uncertain samples were qPCR negative,which may resulted from low PVL in specimens or cross-reactions or non-specific reactions detected by LIA.Therefore,longer-term follow-up and monitoring are urgently needed.In summary,the qPCR method based on Taqman probes can accurately and quantitatively detect the HTLV-1 PVL.In view of its low detection cost,high sensitivity,good specificity,and convenient and fast detection,we expected to apply this method to detect the initially ELISA reactive specimens of the blood donors and then the negative samples were furtherly tested by confirmatory tests.This strategy can not only quantify and type the positive samples,but also reduce the number of western blots or immunoassays.In addition,as the PVL could be a good prognostic marker of related disease development,this method provides an effective way to monitor the PVL of HTLV-1 infections during the long-term follow-up. |
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