OBJECTIVE: To study the hematologic and molecular features of 14 patients with hemoglobin (Hb) variants, so as to provide reference data for its laboratory screening. METHODS: A total of 1 029 samples were screened by high performance liquid chromatography (HPLC) on the Bio-Rad VariantⅡHPLC system. GAP-PCR and reverse dot blot (RDB) were used to detect common mutation of α and β globin gene in Chinese. DNA sequencing for α and β globin gene was simultaneously performed in samples with abnormal spectrum peak and negative thalassemia gene. RESULTS: In 1 029 samples, 10 types of structural Hb variants were detected in14 cases (1.36%), including 1 case of Hb E / β- thalassemia, 1 case of Hb E /α- thalassemia (HbH disease), 2 cases of HbG-Taipei, 2 cases of Hb Q-Thailand, 2 cases of Hb Youngstown, 1 case of Hb Guangzhou-Hangzhou, 1 case of Hb M-Boston, 1 case of Hb G-Siriraj, 1 case of Hb J-Baltimore, 1 case of Hb J-Sicilia and 1 case of Hb Tamano. CONCLUSION The occurrence of abnormal structural Hb variants with many genotypes in Shanghai is unique. Except for Hb E, Hb Youngstown, and Hb M-Boston, other types of heterozygous are normal in phenotypes, and symptoms such as hemolysis and anemia often occur when other diseases are combined.
China ; Genotype ; Hemoglobins, Abnormal/genetics* ; Humans ; Phenotype ; alpha-Thalassemia ; beta-Globins/genetics*

OBJECTIVE: To proceed the clinical evaluation of DNA microarray for thalassemia gene detection. METHODS: Peripheral blood samples of 166 thalassemia gene test subjects were collected and tested for thalassemia genes by microarray chip method and Gap-PCR method combined with PCR-reverse dot blot hybridization method according to double-blind control test. The specificity, sensitivity, positive predictive value, negative predictive value, and total coincidence rate of the microarray chip method were evaluated. When the two methods were inconsistent, multiplex ligation dependent probe amplification (MLPA) was used to verify the deletional α-thalassemia. RESULTS: Compared with Gap-PCR method, specificity, sensitivity, positive predictive value, negative predictive value, Youden index, and total coincidence rate of microarray chip method was 100% (70/70), 96.88% (93/96), 100% (93/93), 95.89% (70/73), 0.969, and 97.59% (162/166), respectively, while compared with PCR-reverse dot blot hybridization method was 100% (125/125), 100% (41/41), 100% (41/41), 100% (125/125), 1, and 100% (166/166), respectively. CONCLUSION The microarray chip method for α-thalassemia gene detection shows the advantages of high specificity, sensitivity, and throughput.
Genetic Testing ; Humans ; Multiplex Polymerase Chain Reaction ; Oligonucleotide Array Sequence Analysis ; alpha-Thalassemia/genetics*

OBJECTIVE: To perform dried blood spots thalassemia gene detection in patients with positive blood phenotypes by microarray technology, and evaluate its value in clinical detection. METHODS: DNA samples were extracted from dried blood spots of 410 patients. Microarray technology was used to detect 3 deletion and 3 non-deletion types of α-thalassemia and 19 β-thalassemia point mutations which were common gene mutions in China. RESULTS: There were 357 positive cases in all the 410 tested samples with the positive rate 87.07%, among which 299 cases (72.93%) carried deletion or point mutations of α-thalassemia, 29 cases (7.07%) carried point mutations of β-thalassemia and 29 cases (7.07%) carried gene mutations of complex αβ-thalassemia syndrome. The mutations of α-thalassemia were involved with -- CONCLUSION The most common genetic mutations are --
China ; Humans ; Mutation ; Oligonucleotide Array Sequence Analysis ; alpha-Thalassemia/genetics* ; beta-Thalassemia/genetics*

OBJECTIVE: To investigate the effect of ursane triterpenoids 3β,19α-dihydroxyursu-12-ene-23,28-dicarboxylic acid (Rotundioic acid, RA) on the sensitivity of adriamycin-resistant K562 cells (K562/ADM Cell) anti-tumor drug, and to explore the effect and mechanism of RA on the multidrug resistance of K562/ADM cells. METHODS: CCK-8 method was used to detect the effect of RA on the sensitivity of K562 cells and K562/ADM cells to anti-tumor drug. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression level of mRNA and the protein in K562 and K562/ADM cells, and the effect of RA on the expression of MDR1 mRNA and P-gp in K562/ADM cells was also detected; Western blot was used to detect the expression of p-JNK, p-p38 and p-ERK1/2 in K562/ADM cells. RESULTS: RA could increased the sensitivity of K562/ADM cells to adriamycin(the reversal factor was 1.61 times), the difference showed statistically significantly (P<0.05); the resistance factor of K562/ADM to ADM was 41.76 times. The expression of MDR1 mRNA in K562 cells was extremely low, and the protein product P-glycoprotein (P-gp) was almost not expressed; MDR1 mRNA and P-gp in K562/ADM cells were highly expressed; RA could down-regulate the expression levels of MDR1 and P-gp in K562/ADM cells. In addition, RA could upregulate the phosphorylation levels of p38 and ERK1/2 in K562/ADM cells, but it has no effect on the expression of p-JNK. CONCLUSION RA may participate in the regulation of MAPK signaling pathway by upregulating the expression levels of p-p38 and p-ERK1/2 in K562/ADM cells, and thus inhibit the transcription and translation levels of MDR1, and finally reverse the multidrug resistance of leukemia cells.
ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1 ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; K562 Cells

OBJECTIVE: To investigate the effect of prostaglandin E recoptor 4 antagonist (EPA) on the self-renewal ability of human CD34 cells and its mechamism. METHODS: The peripheral blood hematopoietic stem cell of 20 healthy donors received the G-CSF-mobilization were collected, then the human CD34 cells were sorted out by MACS microbead kit. The human CD34 cells were treated with DMSO (control group), EPA (EPA group) and EPA+EPA antagonist (EPA+EPA group) for 72 hours. The differential genes and pathways related with CD34 cell stemness were detected by Thermogram and Pathway enrichment analysis. and then the expression levels of protein and gene (β-catenin, Nanog, Oct4, Sox2, Stat3, AKT, P38) were detected by qRT-PCR and Western blot respectively. RESULTS: EPA could elevate the mRNA and protein expression of β-catenin, Nanog, Oct4, Sox2, in comparison with control group, however, mRNA and protein expression of STAT3, AKT, P38 were not changed. When human CD34 cell were cultured with EPA+XAV939 it was found that the mRNA and protein expression of β-catenin was downregulated, moreover the mRNA and protein expression of Nanog, Oct4, Sox2 were reduced. CONCLUSION EPA can upregulate stemness factors-β-catenin, Nanog, Oct4 and Sox2 in human CD34 cell in vitro, but not STAT3, AKT and P38.

OBJECTIVETo investigate the expression of phosphoglycerate kinase 1 (PGK1) and its prognostic value in endometrial carcinoma (EC).

METHODSThe expression of PGK1 was detected immunohistochemically in 30 normal endometrium and 130 EC specimens. The relationship between PGK1 protein expression and the clinicopathological features of the patients was evaluated.

RESULTSImmunohistochemical analysis revealed low PGK1 expression in 55.4% (72/130) and high PGK1 expression in 44.6% (58/130) of the EC specimens, as compared with the rates of 90% (27/30) and 10% (3/30) in normal endometrium, respectively (P<0.001). PGK1 expression was significantly correlated with FIGO stage (P<0.001), histological grade (P=0.002) and lymph node metastasis (P<0.001). Kaplan-Meier survival analysis indicated that patients with a high PGK1 expression had a shorter overall survival rate than those with a low PGK1 expression (P<0.001). Multivariate analysis showed that a high PGK1 expression was not the independent predictor of the prognosis of EC (P=0.077).

CONCLUSIONA high expression of PGK1 is associated with aggressive and metastatic behaviors of EC, and detection of PGK1 provides assistance in evaluating the prognosis of patients with EC.

OBJECTIVETo investigate the effects of minimally invasive esophagectomy (MIE) and open esophagectomy (OE) on the level of circulating tumor cells (CTCs) in patients with esophageal cancer (EC).

METHODSA total of 73 patients with EC undergoing MIE (n=38) or OE (n=35) in our department between October, 2015 and October, 2017 were enrolled, with 10 patients with benign esophagus disease and 10 healthy volunteers as controls. The levels of CTCs in the peripheral blood of the participants were detected using CanPatrol technique and analyzed for their association with the operation methods and perioperative complications.

RESULTSCTCs were detected in 60.3% (44/73) of the EC patients but in none of the control subjects. CTC level after the surgery was significantly higher than that during the surgery, and CTC level during the surgery was significantly higher than that before surgery (P<0.001). The preoperative and intra-operative CTC levels were not significantly different between MIE and OE groups (P>0.05), but the postoperative CTC level was significantly lower in MIE group than in OE group, and postoperative increment of CTC level (from the preoperative level) was significantly lower in MIE group than in OE group (P<0.001). The total incidence of postoperative complications was significantly lower in MIE group than in OE group (28.9% vs 54.3%, P=0.023), and in both groups, CTC levels in patients with complications were significantly higher than those in patients without complications (P=0.001 and P=0.005 in MIE and OE groups, respectively).

CONCLUSIONMIE may help to reduce the number of peripheral blood CTCs early after the operation, and dynamic monitoring CTCs level assists in evaluation of the prognosis of EC patients. CTC level may serve as an indicator for monitoring the prognosis of EC.

OBJECTIVETo explore the long-term prognosis and health-related quality of life of patients surviving hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF).

METHODSThe clinical data were collected from patients with HBV-ACLF, who were hospitalized in our department between November, 2011 and October, 2016 and survived for more than 90 days. The patients were followed for occurrence of newly diagnosed cirrhosis, decompensation events, hepatocellular carcinoma and death. The quality of life of the patients was evaluated using SF-36 score, and the patients with chronic hepatitis B (CHB) and cirrhosis treated during the same period served as controls.

RESULTSA total of 223 ACLF survivors were included in this study. According to the presence of cirrhosis on admission, the enrolled patients were divided into chronic hepatitis B-related ACLF (CHB-ACLF) group (n=130) and liver cirrhosis ACLF (CIR-ACLF) group (n=93). The 12-, 24- and 50-month survival rates in CHB-ACLF group were 97%, 95.7% and 93.9%, respectively, significantly higher than the rates in CIR-ACLF group (91%, 86% and 74%, respectively; P=0.007). In patients with CHB-ACLF, the 12-, 24- and 36-month progression rates of cirrhosis were 37.9%, 58.4% and 68.7% respectively. Multivariate Cox regression identified the peak value of serum creatinine (HR=1.015, P=0.026) and INR (HR=2.032, P=0.006) within 28 days as independent risk factors and serum sodium at baseline (HR=0.84, P=0.035) as an independent protective factor of occurrence of cirrhosis. The score of mental health on SF-36 in ACLF group was significantly lower than the national norms, and the scores for general health and body pain of ACLF patients were significantly higher than those in patients with CHB or cirrhosis.

CONCLUSIONThe long-term prognosis of ACLF survivors with and without cirrhosis can be different. Acute attacks are associated with an increased rate of cirrhosis progression in CHB patients who recovered from ACLF, possibly in relation with the severity of extra-hepatic organ injuries. The physical and social functions of long-term survivors of ACLF do not significantly decline, but their psychological status can be affected.

OBJECTIVETo assess the larvicidal effects of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or Bacillus thuringiensis subsp israelensis (B.t.i) toxin Cyt2Ba against the second instar larvae of Culex pipiensquinquefasciatus and Aedes albopictus and compare different formulations for their larvicidal effects.

METHODSThe AaIT- or Cyt2Ba-coding sequences were cloned into pET28a(+) and the recombinant plasmids were transformed into E. coli BL21(DE3). After induction with IPTG, the recombinant proteins expressed by the recombinant E. coli were detected and identified by SDS-PAGE and Western blotting, respectively. The larvicidal activity of the bacterial suspension was tested at different concentrations against mosquitoes. The effective engineered bacteria were prepared into dry powder with different formulations, and their larvicidal activity was tested.

RESULTSAaIT and Cyt2Ba proteins were successfully expressed in E. coli. The recombinant AaIT protein showed no virulence to the mosquito larvae. The suspension of the recombinant E. coli expressing Cyt2Ba protein exhibited a stronger killing effect on Aedes albopictus larvae than on Culex pipiens quinquefasciatus larvae at 48 h (P<0.001) with LCof 3.00×10cells/mL and 1.25×10cells/mL, respectively. The dry powder of the engineered bacteria formulated with yeast extract, wheat flour or white pepper powder at the mass ratio of 1:1 showed the strongest killing effect on mosquito larvae (P=0.044), and the formulation with white pepper powder produced a stronger killing effect than formulations with yeast extract or wheat flour (P=0.002).

CONCLUSIONThe B.t.i Cyt2Ba protein expressed in E. coli BL21(DE3) shows a good larvicidal activity against mosquitoes, and appropriate formulations of the engineered bacteria can enhance its efficiency in mosquito control.

OBJECTIVETo investigate the effect of monoamine oxidase inhibitor phenelzine on in vitro growth and proliferation of mantle cell lymphoma Jeko-1 cells and its possible mechanism.

METHODSMTT assay was used to observe the cell proliferation and to draw a growth curve. The cell apoptosis was measured by flow cytometry. The expressions of apoptosis-related protein and Wnt signal pathway as well as the level of acetylation of histone were analyzed by Western blot.

RESULTSPhenelzine inhibited proliferation and promoted apoptosis of Jeko-1 cells in a dose-dependent way by increasing the expression of apoptosis related protein BAX, Caspase-3 and p21, while decreasing anti-apoptotic protein BCL-2. In addition, phenelzine could upregulate histone H3K4mel, H3K4me2 and histone acetylated H3, without affecting hitone H3K4me3. Moreover, phosphorylation of GSF-3β, β-catenin, c-myc and cyclinD1 decreased after exposure to phenelzine for 24 hours.

CONCLUSIONPhenelzine can inhibit Jeko-1 cell proliferation and induce apoptosis by regulating methylation and acetylation of histone and suppressing Wnt/β-catenin signal pathway, suggesting its therapeutic benefit for mantle cell lymophma.