Effective suppression of yellow head virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA).

Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block yellow head virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called "pLVX-lhRdRp", against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24-72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.

Investigation of FoxO3 dynamics during erythroblast development in ß-thalassemia major.

The FoxO3 transcription factor is a key regulator of oxidative stress and erythroid maturation during erythropoiesis. In this study, we explored the involvement of FoxO3 in severe ß-thalassemia. Using primary CD34+ hematopoietic progenitor cells from patients with ß-thalassemia major, we successfully developed an in vitro model of ineffective erythropoiesis. Based on this model, FoxO3 activity was quantified in single cells using high throughput imaging flow cytometry. This study revealed a significant reduction of FoxO3 activity during the late stage of erythroblast differentiation in ß-thalassemia, in contrast to erythropoiesis in normal cells that maintain persistent activation of FoxO3. In agreement with the decreased FoxO3 activity in ß-thalassemia, the expression of FoxO3 target genes was also found to decrease, concurrent with elevated phosphorylation of AKT, most clearly at the late stage of erythroid differentiation. Our findings provide further evidence for the involvement of FoxO3 during terminal erythropoiesis and confirm the modulation of the PI3K/AKT pathway as a potential therapeutic strategy for ß-thalassemia.

Enhancement of ß-Globin Gene Expression in Thalassemic IVS2-654 Induced Pluripotent Stem Cell-Derived Erythroid Cells by Modified U7 snRNA.

The therapeutic use of patient-specific induced pluripotent stem cells (iPSCs) is emerging as a potential treatment of ß-thalassemia. Ideally, patient-specific iPSCs would be genetically corrected by various approaches to treat ß-thalassemia including lentiviral gene transfer, lentivirus-delivered shRNA, and gene editing. These corrected iPSCs would be subsequently differentiated into hematopoietic stem cells and transplanted back into the same patient. In this article, we present a proof of principle study for disease modeling and screening using iPSCs to test the potential use of the modified U7 small nuclear (sn) RNA to correct a splice defect in IVS2-654 ß-thalassemia. In this case, the aberration results from a mutation in the human ß-globin intron 2 causing an aberrant splicing of ß-globin pre-mRNA and preventing synthesis of functional ß-globin protein. The iPSCs (derived from mesenchymal stromal cells from a patient with IVS2-654 ß-thalassemia/hemoglobin (Hb) E) were transduced with a lentivirus carrying a modified U7 snRNA targeting an IVS2-654 ß-globin pre-mRNA in order to restore the correct splicing. Erythroblasts differentiated from the transduced iPSCs expressed high level of correctly spliced ß-globin mRNA suggesting that the modified U7 snRNA was expressed and mediated splicing correction of IVS2-654 ß-globin pre-mRNA in these cells. Moreover, a less active apoptosis cascade process was observed in the corrected cells at transcription level. This study demonstrated the potential use of a genetically modified U7 snRNA with patient-specific iPSCs for the partial restoration of the aberrant splicing process of ß-thalassemia. Stem Cells Translational Medicine 2017;6:1059-1069.

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers.

BACKGROUND: The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. RESULTS: The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. CONCLUSION: The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

Molecular, hematological and clinical aspects of thalassemia major and thalassemia intermedia associated with Hb E-beta-thalassemia in Northeast Thailand.

Hb E-beta-thalassemia is the most common form of beta-thalassemia found in Thailand. The disease exhibits a varied clinical expression ranging from severe transfusion dependence to relatively mild thalassemia intermedia. We evaluated the effects of primary and secondary genetic factors in modulating the hematological and clinical presentation of 148 northeast Thai patients including 103 severe thalassemia major (TM) and 45 thalassemia intermedia (TI). Among 148 cases examined, eleven different mutations including two novel ones; (beta(33/34 (-G)) and beta(IVS2#815 C-T)) were identified in trans to the beta(E) gene in two TM cases. The other 9 known mutations included beta(41/42), beta(17), beta(IVS2#654), beta(-28), beta(71/72), beta(35), beta(IVS1#5), beta(IVS1#1) and beta(41). Except for the beta(-28) mutation which was found only in the TI group, others mutations were identified in both TM and TI. Co-inheritance of alpha-thalassemia as a phenotype modulating factor was not evident in this study, nor was the presence of the -158 (G)gamma-globin Xmn I polymorphism. Further analysis of the polymorphic (TG)n(CG)m repeats within the IVS2 of the two gamma-globin genes revealed no different proportions of the polymorphic patterns among TM and TI groups of patients either. Our data reveals that in the majority of these Hb E-beta-thalassemia patients, it is very hard to predict the clinical phenotype of the patients from the beta-globin mutations and these secondary genetic modifiers.

A T-to-G transversion at nucleotide -567 upstream of HBG2 in a GATA-1 binding motif is associated with elevated hemoglobin F.

Increased fetal hemoglobin (Hb F; alpha(2)gamma(2)) production in adults can ameliorate the clinical severity of sickle cell disease and beta-thalassemia major. Thus, understanding the regulation of gamma-globin gene expression and its silencing in adults has potential therapeutic implications. We studied a father and son in an Iranian-American family who had elevated Hb F levels and found a novel T-to-G transversion at nucleotide (nt) -567 of the HBG2 promoter. This mutation alters a GATA-1 binding motif to a GAGA sequence located within a previously identified silencing element. DNA-protein binding assays showed that the GATA motif of interest is capable of binding GATA-1 transcription factor in vitro and in vivo. Truncation analyses of the HBG2 promoter linked to a luciferase reporter gene revealed a negative regulatory activity present between nt -675 and -526. In addition, the T-to-G mutation at the GATA motif increased the promoter activity by two- to threefold in transiently transfected erythroid cell lines. The binding motif is uniquely conserved in simian primates with a fetal pattern of gamma-globin gene expression. These results suggest that the GATA motif under study has a functional role in silencing gamma-globin gene expression in adults. The T-to-G mutation in this motif disrupts GATA-1 binding and the associated repressor complex, abolishing its silencing effect and resulting in the up-regulation of gamma-globin gene expression in adults.