ETS1 and SP1 drive DHX15 expression in acute lymphoblastic leukaemia.

DHX15 plays a role in leukaemogenesis and leukaemia relapse. However, the mechanism underlying the transcriptional regulation of DHX15 in ALL has not been elucidated. Our present study aimed to explore the functional promoter region of DHX15 and to investigate the transcription factors controlling the transcription of this gene. A luciferase assay performed with several truncated constructs identified a 501-bp region as the core promoter region of DHX15. Site-directed mutagenesis, electrophoretic mobility shift and chromatin immunoprecipitation assays showed that ETS1 and SP1 occupied the DHX15 promoter. Furthermore, knockdown of ETS1 and SP1 resulted in suppression of DHX15, whereas the overexpression of these genes led to up-regulation of DHX15. Interestingly, in samples obtained from patients with ALL at diagnosis, both ETS1 and SP1 correlated positively with DHX15 expression. Additionally, differences in methylation of the DHX15 core promoter region were not observed between the patients and controls. In conclusion, we identified the core promoter region of DHX15 and demonstrated that ETS1 and SP1 regulated DHX15 expression in ALL.

[Genetic Analysis of a Chinese Pedigree with Hereditary Spherocytosis Caused by Copy Number Variation Deletion of SPTB Gene].

OBJECTIVE: To investigate the molecular mechanism of the disease based on the clinical characterization and genetic mutation analysis in a family with hereditary spherocytosis. METHODS: The proband with jaundice and anemia was referred to Yidu Central Hospital of Weifang in May 2021. Peripheral blood samples were collected from six members of the family. Second-generation sequencing was used to screen the pathological mutations, and the clinically significant variant sites were selected. Then the relevant databases were used to analyze the variant sites, and RT-qPCR was used to detect the relative mRNA levels of candidate gene. The structure and function of SPTB protein were analyzed by UniProt and SMART databases. RESULTS: We infer that the SPTB gene copy number variation (CNV) deletion was co-segregated with the phenotype of the patients in this family based on the results of second-generation sequencing (about 700 target genes). The UCSC Genome Browser demonstrated that the deleted region was mainly located in exon2-3 of SPTB gene. The results of RT-qPCR showed that the relative SPTB mRNA levels of all patients were lower than the healthy control. UniProt and SMART databases analysis showed that SPTB protein without CH1 and CH2 domains could not bind to erythrocyte membrane actin. CONCLUSION: The CNV deletion of SPTB gene may be the reason for the hereditary spherocytosis in this family.

Primary B-cell non-Hodgkin lymphoma of the parotid gland: An analysis based on the SEER database.

Primary malignant lymphoma of the parotid gland is a rare entity. The disease is often misdiagnosed, and its survival factors remain unclear. This study included patients diagnosed with primary B-cell non-Hodgkin lymphoma of the parotid gland from 1987 to 2016 in the surveillance, epidemiology, and end results program. Univariate survival analysis was conducted using the Kaplan-Meier method, and multivariate analysis was performed using the Cox proportional hazards regression model. A competing risks regression model was applied to estimate the specific risks associated with parotid lymphoma mortality. A total of 1443 patients were identified. The overall survival of indolent primary B-cell lymphoma of the parotid gland was higher than that of aggressive lymphoma (hazard ratio 0.53, 95% confidence interval 0.44-0.64, P < .001), and older patients (≥70 years) exhibited inferior overall survival. Histological subtype and age are important prognostic factors in patients with primary B-cell non-Hodgkin lymphoma of the parotid gland.

A Novel and Rapid Smear Cytomorphology Detection Strategy Based on Upconversion Nanoparticles Immunolabeling Integrated with Wright's Staining for Accurate Diagnosis of Leukemia.

Background: Accurate, sensitive, and rapid identification of leukemia cells in blood and bone marrow is of paramount significance for clinical diagnosis. An integrative technique combining traditional cytomorphology with immunophenotyping was proposed to improve the diagnostic efficiency in leukemia. On account of high photostability, biocompatibility, and signal-to-background ratio, upconversion nanoparticles (UCNPs) as luminescent labels have drawn substantial research scrutiny in immunolabeling. Methods: To achieve simultaneous determination, NaYF4:Yb,Er UCNPs were coupled with CD38 antibodies to construct immunofluorescence probes that were developed to bind to diffuse large B cell lymphoma (DLBCL) cells, followed by Wright's staining that has been widely used in clinical work for morphological diagnosis. Further, the experimental conditions were optimized, such as medium, slice-making method, antibody dosage, incubation time, etc. Results: The cell morphology and immunolabeling could be observed simultaneously, and its simple operation rendered it a possibility for clinical diagnosis. The developed immunolabeling assay could achieve DLBCL cell counting with high reproducibility and stability, and the detection limit was as low as 1.54 cell/slice (>3 σ/s). Moreover, the proposed method also realized real blood and bone marrow sample analysis, and the results were consistent with the clinical diagnosis. Conclusion: Overall, this strategy can be carried out after simple laboratory training and has prospective biomedical applications in leukemia classification, diagnosis validation, and differential diagnostics.

The prognostic value of the peripheral blood cell counts changes during induction chemotherapy in Chinese patients with adult acute myeloid leukemia.

ABSTRACT: To investigate the prognostic value of the circulating peripheral blood cell counts changes in acute myeloid leukemia (AML) at different time points during induction chemotherapy.We retrospectively analyzed the clinical and laboratory data of 237 newly diagnosed AML patients admitted to Fujian Medical University Union Hospital from January 2011 to December 2014.1. When primitive cells were first removed from the circulating peripheral blood, it was called peripheral blood blast clearance (PBBC). These patients were divided into two groups, according to PBBC. Statistical analysis showed that the day 5 of induction chemotherapy was a better cut-off for PBBC. PBBC≤5 days is defined as early-blast-clearance, while PBBC >6 days is delayed-blast-clearance. There was significant difference between the two groups on complete remission (CR) rate (P = .002), recurrence-free survival (RFS) (P = .026) and overall survival (OS) (P = .001). 2. Multivariate analysis suggested PBBC is an independent prognostic factor for CR, RFS, and OS in AML. Receiver operating characteristic(ROC) curve analysis showed the CR rate of patients with white blood cell count less than 1.25 × 109/L was significantly higher than that of patients with white blood cell count more than 1.25 × 10 9/L (P < .001) at day 5 of induction chemotherapy, but the RFS and OS was no significantly different (P > .05).The dynamics of peripheral blood blast in AML after initiation of induction chemotherapy, especially the time length to achieve PBBC, has important prognostic value for CR rate, RFS, and OS in AML patients. It is a simple and feasible method to evaluate the efficacy of AML.

[Efficacy and Safety of Decitabine Combined with CAG (Cytarabine, Aclarubicin, G-CSF) for Patients with Intermediate or High Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia: a Meta-Analysis].

OBJECTIVE: To systematically evaluate the efficacy and safety of DCAG regimen for treating the intermediate or high risk MDS and AML. METHODS: PubMed, EMbase, The Cochrane Library, WanFang Data and CNKI databases were searched to collect randomized controlled trials (RCTs) of decitabine combined with CAG regimen for intermediate or high risk MDS and AML from inception to March, 2018. The quality of each RCT was evaluated by the Cochrane collaboration´s tool for assessing the risk of bias.Then, the data were analyzed by using RevMan 5.3. RESULTS: Twenty-four RCTs were included in the meta-analysis, containing 1 557 patients with intermediate or high-risk MDS and AML, of whom 594 were AML patients and 590 were MDS patients. The patients treated with the DCAG regimen were enrolled in DCAG group, and the patients treated with single-agent decitabine or CAG regimen were enrolled in control group. RESULTS: The results of meta-analysis showed that compared with other therapies, the complete remission rate of DCAG regimen in patients with intermediate or high-risk MDS and AML was high (RR=1.63，95% CI=1.43-1.85，P＜0.000 01), and the overall response rate was also high (RR=1. 35，95% CI=1.24-1.46，P＜0.000 01); Subgroup analysis results showed that DCAG regimen was better than CAG regimen in the complete remission rate (RR=1.71，95% CI=1.49-1.97，P＜0.000 01), and slightly better than single-agent decitabine group (RR=1.43，95% CI=1.08-1.91，P=0.01). In terms of adverse reactions, there was no statistically significant difference in the rates of myelosuppression, pulmonary infection, gastrointestinal reactions, and bleeding events between the 2 groups (P＞0.05). CONCLUSION: DCAG regimen has significant efficacy in the treatment of intermediate or high-risk MDS and AML, and is superior to CAG regimen and single-agent dicitabine regimen. As compared with control group, there was no significant difference in adverse events. Due to limited quantity and quality of the included studies, more high quality studies are needed to verify above mentioned conclusion.

[Full-length cDNA cloning and biological function analysis of a novel gene FAMLF related to familial acute myelogenous leukemia].

OBJECTIVE: To clone the full-length cDNA of a novel gene related to familial acute myelogenous leukemia (AML) and to demonstrate its molecular mechanisms on the gene level. METHODS: Bone marrow specimen was obtained from a patient of familial AML, male, aged 11, and peripheral blood samples were obtained from 23 AML patients outside this family, 9 normal persons in this family, and 23 normal persons outside this family. Based on the EST sequence zywb87 (GenBank accession number: CV973101) from a subtractive cDNA library of differential expressed genes constructed in familial AML, SMART-rapid amplification of cDNA ends (SMART-RACE) was applied to clone the full-length cDNA of the novel gene, and bioinformatics was used to predict its biological function, the expression of the novel gene in AML was detected by One-Step RT-PCR. RESULTS: A full-length cDNA of 2313 bp was obtained from the bone marrow specimen of the familial AML patient with complete open reading frame (ORF) of 249 bp. Localized on 1q31.3 of human chromosome, it coded a 82-amino acid polypeptide with signal peptide, leucine-rich repeat (LRR_SD22), and intrinsic disorder functional domain. BLAST analysis confirmed this gene as a novel gene designated with the accession number: (nucleotide) EF413001 and (protein) ABN58747 by GenBank and was named as Homo sapiens familial acute myelogenous leukemia related factor (FAMLF). The FAMLF expression level of the AML patients outside this family was (2.61 +/- 0.66), significantly higher than that of the normal persons outside this family (0.97 +/- 0.51, P < 0.01). CONCLUSION: A full-length cDNA of the novel gene FAMLF related to familial AML has been obtained. The FAMLF gene is expressed highly in AML and may present biological function on the progress of AML.

[Full-length cloning of a novel gene, ELF2C, related to familial acute myelogenous leukemia].

OBJECTIVE: To clone the full-length cDNA of a novel gene of familial acute myelogenous leukemia and to explain the molecular mechanism of the disease at the gene level. METHODS: Based on a EST sequence (zywb4) from a subtractive cDNA library of specially or differentially expressed genes constructed in familial acute myelogenous leukemia, electronic cDNA cloning and SMART-rapid amplification of cDNA ends (SMART-RACE) were used to clone the full-length cDNA of a novel associated gene of familial acute myelogenous leukemia. RESULTS: One sequence of 2257 bp was obtained, which obeyed Kozak rules, and contained an open reading frame (ORF) and a polyA tail. BLAST analysis showed that it may be a novel gene. The new sequence was submitted to GenBank with the accession number: DQ359746 and designated as ELF2C. CONCLUSION: A full- length cDNA of a novel gene (ELF2C) related to familial acute myelogenous leukemia is obtained, which is helpful to further investigation of its function in familial acute myelogenous leukemia.

Lentivirus-mediated RNA interference targeting FAMLF-1 inhibits cell growth and enhances cell differentiation of acute myeloid leukemia partially differentiated cells via inhibition of AKT and c-MYC.

Genetic heterogeneity is the basis of clinical heterogeneity among different subtypes of AML. We have successfully cloned a gene related to AML termed FAMLF from a FAB-M2 patient's sample of a second largest AML pedigree. Then we revealed at least three splice variants, named as FAMLF-1, FAMLF-2 and FAMLF-3, and found miR181a1/b1 in the second intron of FAMLF gene family. Higher expression of FAMLF-1 was related to a higher complete remission (CR) rate, but shorter relapse free survival (RFS) in AML. We further found that the FAMLF-1 single nucleotide polymorphism (SNP) haplotype and its expression were positively correlated to clinical parameters of acute myeloid leukemia partially differentiated (FAB-M2) patients, but not FAB non-M2 patients or Acute Monocytic Leukemia (FAB-M5) patients. GTAGG SNP haplotype of FAMLF gene might increase FAB-M2 susceptibility in Han population and act as a useful candidate biomarker for FAB-M2 screening. We also demonstrated that FAMLF-1 gene silencing in FAB-M2 cells could lead to proliferation inhibition, cell cycle G0/G1 phase arrest, and differentiation promotion independent of its intronic miR-181a1, which might be related to Akt/c-Myc pathway. These findings reveal a role of FAMLF-1 as a potential pathogenic gene for FAB-M2.

Influences of the Three Gorges Project on seismic activities in the reservoir area.

Reservoir-induced earthquakes related with the construction of the Three Gorges Project have attracted great concerns of the public. Since the first water impoundment on May 25, 2003, a number of earthquakes have occurred during the water storage stages, in which the largest was the Badong M5.1 earthquake on December 16, 2013. In this paper, the relationships between seismic activities, b value, seismic parameters, and reservoir water level fluctuations are studied. In addition, based on the digital seismic waveform data obtained since 2000, the focal depth changes and focal mechanism characteristics before and after the water impoundment are studied as well. These provide us important information to understand the earthquake mechanisms. The results show that these earthquakes are typical reservoir-induced earthquakes, which are closely related to water infiltration, pore pressure, and water level fluctuations. The majority of the micro and small earthquakes are caused by karst collapse, mine collapse, bank reformation, superficial unloading, and so on. The larger earthquakes are related to the fault structures to some extent. Due to the persistent effects of water impoundment on the seismic and geological environments around the reservoir and water infiltration into the rocks, the influences on the crustal deformation field, gravity field, seepage field, and fault medium-softening action may vary gradually from a higher strength to a weaker one. Therefore, it is possible that small earthquakes and few medium earthquakes (M≤5.5) will occur in the reservoir area in the future.

[Establishment and evaluation of oxidative stress injury model of in vitro rat aortic endothelial cells].

OBJECTIVE: To establish a model of oxidative stress injury in cultured rat aortic endothelial cells, and to provide a basis for the research of cell injury and apoptosis. METHODS: The rats were decapitated to get the aorta in thoracic operation under aseptic conditions. By subculture after tissue block culture method to get sufficient aortic endothelial cells, cultured in 96-well plates or grow on cover glass for the following test. Without H2O2 group as a control group, with different doses of H2O2 (100,200,300,400,500 µmol/L) treated endothelialcells in 12 h to screen the optimal dose. Based on the results, with the same dose of H2O2 (100 or 200 µmol/L) acted on endothelial cells respectively in different time (3, 6, 9, 12 and 24 h) to screen the optimal duration. Each group was made in sextuplicate. The establishment of the model was evaluated by immunofluorescence,cell viability testing, biochemical indicators detection (lactate dehydrogenase(LDH), nitric oxide(NO), malondialdehyde(MDA), superoxidedismutase(SOD))and apoptosis index testing. RESULTS: Endothelial cells were cultured successfully and verified by immunofluorescence staining of intracellular antigen Ⅷ collagen. With the increase of H2O2 doses at the same action time 12 h, the cell viability was significantly decreased (77.63%±5.20% to 40.90%±2.10%). The same dose(100 µmol/L group and 200 µmol/L group)with the action time increasing, the cellviability was significantly decreased (100 µmol/L group was 86.83%±12.11% to 44.26%±5.70%, 200 µmol/L group was 78.28%±11.98% to 34.45%±5.87%). At dose of H2O2 was 100 µmol/L and treated in 3,6,9,12 and 24 h, LDH-L and MDA were significantly increased after 9 h while NO and SOD were significantly decreased. In H2O2 dose of 100 µmol/L and action time 12 h, flow cytometry showed endothelial cellapoptosis rate was 16.92%±2.37%, significantly higher than the control group of 2.68%±0.47%(P<0.01); TUNEL detected endothelial cell apoptosis index was17.65%±2.36%, which was significantly higher than that in the control group of 3.23%±0.57%(P<0.01). CONCLUSIONS: The method was successfullyestablished a model of oxidative stress injury in cultured rat aortic endothelial cells, explore the moderate conditions that induced cells injury and apoptosis which could be a basis for the research.

[Proliferation promotion and apoptotic inhibition effects of ribosomal protein RPL36A small interference RNA on U937 cells].

This study was purposed to investigate the expression of RPL36A (ribosomal protein 36a) in the newly diagnosed acute myeloid leukemia (AML) cells and its mechanism at the molecular level. The RPL36A mRNA expression in the newly diagnosed AML cells, U937 cells and normal MNCs was determined by RT-PCR. Small interfering RNA (siRNA) targeting to RPL36A was transfected into U937 cells by Lipofectamine 2000 system. Proliferation, cell cycle, apoptosis of U937 were observed through MTT assay, flow cytometry, acridine orange/ethidium bromide (AO/EB) double staining, TUNEL and Annexin V/FITC respectively. RPL36A mRNA and protein expression levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis respectively. The results showed that RPL36A expression in the newly diagnosed AML cells and U937 cells was significantly upregulated. The average OD value of U937 cells transfected with RPL36A siRNA was significantly lower as compared with 3 control groups. The cell percentage in G2-and S-phase increased, which indicated the inhibition effect of RPL36A siRNA on cell proliferation. Remarkable cell apoptosis in U937 cells treated with RPL36A siRNA was observed by AO/EB, TUNEL analysis and Annexin V/FITC assay; RPL36A mRNA and protein expression level of U937 cells treated with siRNA were significantly declined in a time-dependent manner (r=0.9813 and 0.9537). It is concluded that the RPL36A expression in the AML cells is significantly enhanced and the RPL36A gene may be involved in regulation of cell cycle and cell apoptosis of AML, which promotes proliferation of AML cells and inhibits apoptosis of cells.