ABSTRACT
Objective@#To clarify the prevalence, clinical features and molecular characteristics of germline GATA2 mutations in pediatric primary myelodysplastic syndromes (MDS) .@*Methods@#Next-generation sequencing technology was used to detect mutations in GATA2 and other myeloid malignancy genes in 129 children with primary MDS from Jan. 2007 to Jan. 2018. The relationship between genotypes and phenotypes was analyzed.@*Results@#Germline GATA2 mutations accounted for 8.5% (11/129) of all primary MDS cases, and 14.0% (11/50) of MDS with excess blasts (MDS-EB) and acute myeloid leukaemia with myelodysplasia-related changes (AML-MRC) . Compared with GATA2 wild-type patients, GATA2 mutated patients were older at diagnosis[8 (1-16) years old vs 6 years old (range: 1 month old-18 years old) , P=0.035]and higher risk of monosomy 7 (72.7%vs 5.2%, P<0.001) and classified into MDS-EB and AML-MRC compared with refractory cytopenia of childhood (RCC) (63.6%vs 36.4%, P=0.111) . The multivariate analysis showed SETBP1 mutation (P=0.041, OR=9.003, 95%CI 1.098-73.787) and isolated monosomy 7 (P=0.002, OR=24.835, 95%CI 3.305-186.620) were significantly associated with germline mutated GATA2. Overall survival (OS) and outcomes of hematopoietic stem cell transplantation (HSCT) were not influenced by GATA2 mutational status.@*Conclusions@#Our data identify germline GATA2 mutations have a high prevalence in older pediatric patients with monosomy 7, and high risk of progression into advanced MDS subtypes. GATA2 mutation status does not affect OS in pediatric primary MDS.
ABSTRACT
To study the biological function of DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) gene, we constructed human stable U2OS cell line of DNAH2 gene knockout through CRISPR/Cas9n double nick system. The A, B sgRNAs (Single guide RNA) and complementary strands were designed and synthesized. The double-stranded structures were formed during annealing, and connected with BbsⅠ cohesive ends-containing pX462 linear vector to construct the recombinant eukaryotic expression plasmids, including pX462-DNAH2-A and pX462-DNAH2-B. After the co-transfection of the two plasmids into U2OS cells, the addition of puromycin and limiting dilution method were used to obtain positive monoclonal cell line. Western blotting assay was then performed to detect the expression of DNAH2 protein, and PCR-sequencing technology was finally utilized to analyze the mutation feature. The results showed that A, B sgRNAs duplex was successfully inserted into pX462 vector, and DNAH2 protein was not expressed and DNAH2 gene suffered from the frame-shift mutation in U2OS-DNAH2-KO monoclonal cell line. These demonstrated that DNAH2 knockout U2OS stable cell line was successfully constructed through CRISPR/Cas9n double nick system, which providing a useful tool for the study of DNAH2 gene.
ABSTRACT
Objective To prepare specific mouse monoclonal antibodies against Homo sapiens dynein,axonemal, heavy chain 2 (DNAH2). Methods Firstly, recombinant plasmid encoding His tagged immunogen, targeting N-terminal sequence of DNAH2 protein (1-300 aa), in E. coli was constructed. IPTG was used to induce the expression of His-immunogen, which was then purified and immunized in BALB/c mice. Hybridoma cells were obtained through the fusion between myeloma cells and splenocytes isolated from BALB/c mice. Finally, ELISA and Western blot assays were performed to screen the positive hybridoma. Results IPTG was used efficiently to induce the expression of DNAH2 immunogen in E. coli. DNAH2 protein bands were detected in screened positive hybridoma. Conclusion Mouse monoclonal anti-DNAH2 antibody is prepared successfully.
ABSTRACT
BACKGROUND:Impaired balance between osteogenesis and adipogenesis of bone marrow mesenchymal stem cels is a crucial pathological mechanism of osteoporosis. Mechanical loads applied to bone tissue can increase bone formation and improve bone strength, and meanwhile lead to the release of extracelular nucleotides, such as adenosine triphosphate. OBJECTIVE: To determine the effects of adenosine triphosphate on the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cels and to investigate the underlying mechanisms. METHODS:The effect of adenosine triphosphate (10, 50, 250 μmol/L) on differentiation of bone marrow mesenchymal stem cels were measured by osteogenic and adipogenic related genes expression, alizarin red staining and oil red O staining. The activation of ERK1/2 signaling pathway by adenosine triphosphate was tested using western blot assay. RESULTS AND CONCLUSION: Incubation of bone marrow mesenchymal stem cels with adenosine triphosphate resulted in the dose-dependent increase of osteogenic genes expression and calcium deposition, and inhibition of adipogenic genes expression and lipid droplet formation, but had no effects on cel proliferation. Adenosine triphosphate activated ERK1/2 signaling pathway, and U0126 as an ERK1/2 inhibitor restrained the effect of adenosine triphosphate on the differentiation of bone marrow mesenchymal stem cels.
ABSTRACT
<p><b>OBJECTIVE</b>To establish the ADAR1 (adenosine deaminase that act on RNA 1) knockout MLL-AF9 acute myeloid leukemia (AML) mouse model, and to preliminarily investigate the effects of ADAR1 deletion on the development of AML.</p><p><b>METHODS</b>The lineage⁻ (Lin⁻) cells of ER-CreADAR1(lox/lox) mice and their ADAR1(lox/lox) counterparts were enriched by magnetic activated cell sorting (MACS) and then transduced with retrovirus carrying MSCV- MLL/AF9-IRES-GFP fusion gene. The efficiency of transduction was detected by flow cytometry, and equal number of GFP⁺ cells were transplanted into lethally irradiated recipient mice. The recipient mice were treated with tamoxifen at 48 hours after transplantation to induce ADAR1 knockout and divided into following groups: experimental group (ER-Cre;ADAR1(lox/lox)+tamoxifen), control groups ((1)ER-Cre;ADAR1(lox/lox)+vechile, (2)ADAR1(lox/lox)+tamoxifen, (3)ADAR1(lox/lox)+vechile). The percentage of GFP⁺ cells in peripheral blood was examined at 10, 15 and 20 days respectively after transplantation and the survival of the recipient mice was observed. In vitro study, ER-Cre;ADAR1(lox/lox) and ADAR1(lox/lox) AML cells were cultured and the apoptosis rates of these cells 48 hours after 4-hydroxytamoxifen treatment were examined.</p><p><b>RESULTS</b>The ADAR1 deletion MLL-AF9 AML mouse model was successfully established. Deletion of ADAR1 could decrease the percentage of GFP⁺ cells in the peripheral blood and significantly prolong the survival rate of recipient mice（P<0.05）. In vitro study showed that the cultured total cell number, percentage of GFP⁺ cells decreased and the apoptosis rate of AML cells increased.</p><p><b>CONCLUSION</b>Ablation of ADAR1 could delay the progression of AML in recipient mice. ADAR1 plays a critical role in the development and maintenance of murine MLL-AF9 AML.</p>
Subject(s)
Animals , Mice , Adenosine Deaminase , Apoptosis , Disease Models, Animal , Leukemia, Myeloid, Acute , Myeloid-Lymphoid Leukemia Protein , TamoxifenABSTRACT
Herpes simplex viruses (HSV-1 and HSV-2) cause global morbidity and synergistically correlate with HIV infection.HSV exists life-long in a latent form in sensory neurons with intermittent reactivation,in despite of host immune surveillance.While abundant evidence for HSV interfering with innate immune responses so as to favor the replication and propagation of the virus,several lines of evidence declare that HSV attenuates adaptive immunity by various mechanisms,including but not limited to the ablation of antigen presentation,induction of apoptosis,and interruption of cellular signaling.In this review,we will focus on the perturbative role of HSV in Tcells signaling.