[Using next generation sequencing technology to analyze gene mutations in patients with acute myeloid leukemia and the impact on prognosis].

Objective: To analyze the distribution of gene mutations in newly diagnosed acute myeloid leukemia (AML) patients, based on next generation sequencing technology (NGS) and to evaluate their value in AML risk stratification. Methods: The study analyzed 453 newly diagnosed AML(excluded acute promyelocytic leukemia, APL) patients from seven hospitals in Shanghai, from January 1st 2014 to December 31th 2017. RNA and DNA were extracted from pretreatment bone marrow mononuclear cells and targeted sequencing of AML genes were performed. The data of different groups was compared. Results: A total of 453 newly diagnosed AML patients were enrolled in the study, including 247 males and 206 females with a median age of 49.5 (range,11-85) years. A total of 540 mutations/fusion genes were detected in 289 patients, 29.1% (132/259) of whom with two or more mutations/fusion genes. In all patients, NPM1 was the most common mutation(12.8%), followed by ETO and TET2 mutation (11.92% and 11.04%, respectively) . And WT1 over-expression accounted for 10.6%. Patients over the age of 50 were with a higher frequency of mutations associated with epigenetic modification, 11.93% for ASXL1, 13.99% for DMNT3A, 6.58% for IDH1/IDH2, and 13.17% for TET2. The frequency of DMNT3A mutations was three times higher than that of patients under 50 years of age (P=0.017). In this study, a relatively low proportion of genetic mutations was observed in low-risk karyotype group. In the medium-risk karyotype group, the relatively high mutation frequencies were observed in NPM1, TET2, FLT3-ITD, DNMT3A, ASXL1, and CEBPA genes. In the poor-risk karyotype group, the mutation frequencies of ASXL1, TET2, DNMT3A and PHF6 genes were more than 10%, especially ASXL1 and PHF6 mutation frequencies were significantly higher than other molecular risk stratification groups (P<0.05). Of the 254 patients (56%) with normal karyotype AML (NK-AML), 56 patients were detected to have gene mutations about epigenetic modification. The median OS of this group was worse than that of patients without related mutations, while the median LFS had no significant difference. In patients with NK-AML older than 50 years, the OS and LFS of patients with epigenetic modification related gene mutations was 12 months and 10 months, versus 18 months and 12 months of patients without mutations. Conclusions: The gene mutations frequencies in AML patients with different age and molecular risk stratification groups are different. Epigenetics gene mutation frequencies, such as DNMT3A, ASXL1, IDH1/IDH2 and TET2,are higher in patients older than 50 years. A shorter OS can be observed in older patients(>50 years) with epigenetics gene mutation.

MiR-34a promotes myocardial infarction in rats by inhibiting the activity of SIRT1.

OBJECTIVE: To investigate the effect of micro ribonucleic acid (miR)-34a regulating silent information regulator 1 (SIRT1) on myocardial infarction (MI) rats. MATERIALS AND METHODS: A total of 30 male, 8-week-old rats were divided into three groups, including: sham group (M group), MI group and MI + miR-34a treatment group (miR group). Tissue morphology in the MI region was observed via hematoxylin-eosin (HE) staining. Myocardial apoptosis in the three groups was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Furthermore, the protein levels of SIRT1, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in myocardial cells were detected via Western blotting. RESULTS: Compared with M group, left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) increased significantly in MI group and miR group (p<0.05), while left ventricular ejection fraction (LVEF) and fractional shortening (FS) decreased obviously (p<0.05). The results of HE staining showed that the inflammatory infiltration of myocardial cells and intercellular collagen fibers significantly increased, and the neuronal damage was remarkably aggravated in MI group and miR group when compared with M group (p<0.05). Compared with MI group, myocardial necrosis, inflammatory cell infiltration and intercellular collagen fibers all increased significantly in miR group (p<0.05). Moreover, the results of TUNEL assay revealed that myocardial apoptosis rate in MI group [(21.35±3.12)%] was remarkably higher than that of M group [(9.53±1.17)%]. Meanwhile, it was significantly higher in miR group [(42.38±3.44)%)] than that of MI group, displaying statistically significant differences (p<0.05). The number of apoptotic cells increased obviously in MI group when compared with M group, while it decreased significantly in MI group when compared with miR group (p<0.05). Besides, the protein levels of SIRT1 and Bcl-2 in myocardial tissues in miR group were remarkably lower than those of M group and MI group (p<0.05). Furthermore, the protein level of Bax in miR group was higher than that of M group and MI group, and there were statistically significant differences (p<0.05). CONCLUSIONS: Overexpression of miR-34a inhibits the activity of SIRT1, thereby promoting the apoptosis of MI.

[Expression profiles of microRNA related to atherosclerosis in patients with OSA].

Objective:Recent studies have demonstrated that obstructive sleep apnoea(OSA) may lead to atherosclerosis(AS), but the underlying mechanism remains unclear. MicroRNA(miRNA) may be involved in the gene regulation of AS and also in the occurrence and progression of OSA. The purpose of our study was to investigate the expression of atherosclerosis related serum miRNAs in OSA patients.Method: We screened 116 participants including normal controls(n=24), OSA alone(n=32),OSA patients with AS(n=32),and patients with AS but without OSA(n=28).qRT-PCR to analyze the microRNA profile was performed in these subjects.Clinical and blood test with lipid parameters were analysed using Spearman correlations.Result:Compared to normal controls, miRNA-148a-5p, miRNA-378c, miRNA-127-3p and miRNA-365a-3p were upregulated in the OSA, OSA-AS, and AS groups; miRNA-134-5p was only upregulated in the AS group, miRNA-365a-3p in the OSA-AS group was higher than that in the OSA alone group. The circulating atherosclerosis related miRNAs, including miRNA-148a-5p, miRNA-378c, miRNA-127-3p, miRNA-134-5p and miRNA-365a-3p all correlated with the carotid intima media thickness. Conclusion: Upregulation of miRNA-148a-5p, miRNA-365a-3p, miRNA-378c, miRNA-127-3p may be an early warning marker of atherosclerosis in OSA patients.

[Clinical analysis of 24 cases of aortic dissection during pregnancy].

Objective: To investigate the management and perinatal outcome of aortic dissection during pregnancy. Methods: 24 pregnant women with aortic dissection who delivered in Beijing Anzhen Hospital Affiliated to Capital Medical University from January 1st, 2006 to February 29th, 2016 were recruited. The clinical data, the management and the perinatal outcome were analyzed retrospectively. Results: (1) Clinical features: The average age of the patients was (29±4) years old. The clinical symptoms occurred from 5 gestational weeks to 1 month after delivery. The major symptoms were chest pain, and some complained migratory or radiating pains. There were 17 Stanford type A cases and 7 type B cases. The aortic widths were 2.9-10.0 cm, with the average of (5.6±1.7) cm. D-dimer level was 448-6 421 µg/L, with the average of (1 097±1 209) µg/L, and the fibrin degradation products were 4.1-52.1 mg/L, with the average of (10.2±9.5) mg/L.The white blood cell ranged (6.8-36.4)×10(9)/L, with the average of (29.4±4.2)×10(9)/L; and the neutrophil rate was 76.0%-97.6%, with the average of (84.6±6.3) %. (2) The treatment: ①19 patients underwent aorta surgeries. The surgeries included aortic root replacement with total aortic arch replacement plus stented elephant trunk implantation (Bentall+Sun's surgery), aortic root replacement (Bentall surgery), stent implantation, thoracic and abdominal aorta replacement. The aortic operation time of the 19 patients were 5 gestational weeks to 1 month after delivery. The relation between aortic operation and the termination of pregnancy: 4 patients underwent aorta surgery after termination of pregnancy, 9 patients had cesarean section and aorta surgery at the same time, 6 patients underwent aorta surgery before cesarean section. ② 5 patients did not receive arota surgery, 2 patients of type A dissection and 1 patient of type B dissection died before the surgery; 2 cases of type B dissection underwent conservative treatment. The termination time of pregnancy was 6-37 gestational weeks, with the average of (26±10) weeks. (3)Maternal and fetal outcomes: 20 patients survived after treatment (83%, 20/24) and 4 patients died (17%, 4/24). 10 cases were live births, including 4 full-term infants and 6 preterm premature infants. The birth weight of the neonates was 1 080-3 800 g, with the average of (2 302±764) g. Three of them were very low birth weight infants and 1 was low birth weight infant; 3 neonates had mild asphyxia. The neonates were followed up for 0.5 to 10 years, with the average time of (1.4±1.7) years. So far the infants' development was good. Conclusions: Pregnancy with aortic dissection is pernicious. Early identification, prompt diagnosis and prompt interventing of the vascular surgery are necessary to the safety of mother and fetus.

Overexpression of G100S mutation in PRKAG2 causes Wolff-Parkinson-White syndrome in zebrafish.

The Wolff-Parkinson-White (WPW) syndrome was believed to be associated with PRKAG2 gene mutations. In this study, we verified the pathopoiesis of G100S mutation, a novel mutation only discovered in Chinese patients with WPW, in cardiac disorder. Similar to R302Q, when overexpressed PRKAG2 G100S mutant in zebrafish, we observed a thicker heart wall, detected a decreased AMPK enzymatic activity by tissue AMPK kinase activity colorimetric technique, as well as examined an increased glycogen storage in heart wall using the method for periodic acid-Schiff staining, in comparison with the zebrafish without exogenous PRKAG2 (mock) or with wild-type PRKAG2 (WT). Taken together, we concluded PRKAG2 G100S mutation might contribute to impair the AMP-activated protein kinase function, which resulted in increased cardiac glycogen storage, serving as a pathogenesis for WPW syndrome in Chinese.

Naloxone inhibition of lipopolysaccharide-induced activation of retinal microglia is partly mediated via the p38 mitogen activated protein kinase signalling pathway.

OBJECTIVES: To investigate the effects and underlying mechanism of action of naloxone on lipopolysaccharide (LPS)-induced activation of retinal microglia in vitro. METHODS: Rat retinal microglia primary cultures were divided into four treatment groups: untreated; 1 µg/ml LPS for 12 h; 0.5, 1.0 or 2.0 µM naloxone for 30 min before LPS; 2.5 or 5.0 µM SB203580 for 12 h before LPS and naloxone. Levels of tumour necrosis factor (TNF)-α and interleukin (IL)-1ß were determined by enzyme-linked immuno sorbent assay. Western blot analysis and double immunofluorescence were used to examine activation of the mitogen activated protein kinase (MAPK) signalling pathway. RESULTS: LPS induced an increase in TNF-α and IL-1ß in culture supernatants, which was dose-dependently inhibited by naloxone. Naloxone also dose-dependently inhibited LPS-induced increases in phosphorylated p38 MAPK. Pretreatment of cells with SB203580 attenuated the inhibitory effect of naloxone on TNF-α and IL-1ß production. CONCLUSIONS: Naloxone suppressed LPS-induced activation of cultured retinal microglia and this suppression appeared to occur partly through the p38 MAPK signalling pathway. Naloxone may have therapeutic potential in neuro degenerative diseases characterized by the activation of microglia.

Dynamics of Sept4 expression in fibrotic livers of mice infected with Schistosoma japonicum.

n order to investigate the dynamics of Septin4 (Sept4) expression and its function in the formation of fibrotic livers in mice infected with Schistosoma japonicum, we constructed the mouse model of S. japonicum egg-induced liver fibrosis for 24 weeks. Immunohistochemical staining, qRT-PCR and Western blot were used to detect the expression of Sept4 and α-smooth muscle actin (α-SMA). We found Sept4 localized in the perisinusoidal space where hepatic stellate cells (HSCs) distribute in the periphery of circumoval granulomas and the portal venule. The expression of Sept4 and α-SMA had a similar significant tendency of an up-regulation to a peak at 12 weeks post-infection (p.i.) followed by a down-regulation. At 24 weeks p.i. both were at a low level. These results suggest that Sept4 and α-SMA may interact together in HSCs. Based on this evidence, we hypothesize that Sept4 seems to be involved in the formation of inflammatory granulomata and subsequent liver fibrosis by regulating HSCs activation.

Serum resistin level in essential hypertension patients with different glucose tolerance.

AIMS: To investigate resistin concentrations in patients with essential hypertension and different glucose tolerance and the relationship between serum resistin level and blood glucose. METHODS: Sixty-five patients with essential hypertension [13 with Type 2 diabetes mellitus (DM), 26 with impaired glucose tolerance (IGT), and 26 with normal glucose tolerance (NGT); 30 males, 35 females] were studied. Fasting serum resistin concentrations were measured by enzyme immunoassay (EIA). Oral glucose tolerance tests and insulin release tests were used to calculate glucose area under the curve (AUCG), the ratio of insulin to glucose (DeltaI30/DeltaG30), and insulin sensitivity index (ISI) according to Cederholm's formula. RESULTS: Fasting serum resistin concentrations (microg/l) in DM (34.9 +/- 10.2) patients were significantly higher than those in IGT (25.1 +/- 10.4) (P < 0.05) and in NGT (21.5 +/- 7.9) (P < 0.05) patients. Pearson correlation showed that fasting serum resistin concentration was correlated with AUCG (r = 0.445, P < 0.001), ISI (r = -0.322, P < 0.01) and DeltaI30/DeltaG30 (r = -0.366, P < 0.01), but not body mass index and waist-hip ratio. After adjustment for gender, age and body mass index (BMI), partial correlation analysis showed that the fasting serum resistin concentrations were still correlated with AUCG (r = 0.327, P < 0.01) and DeltaI30/DeltaG30 (r = -0.348, P < 0.01), but ISI. CONCLUSION: Resistin may be involved in the development of diabetes in humans.

[Effect of ICAM-1/LFA-1 on hypoxia/reoxygenation injury of cardiomyocytes mediated by neutrophils].

AIM: To observe the effect of hypoxia/reoxygenation on the adherence of neutrophils to cardiomyocytes and to investigate the effect of ICAM-1/LFA-1 on hypoxia/reoxygenation injury of cardiomyocytes mediated by neutrophils. METHODS: Count adhered neutrophils to cardiomyocytes suffering hypoxia, hypoxia/reoxygenation and normal culture, as well as adhered neutrophils that were blocked by anti ICAM-1 and LFA-1 monoclonal antibodies. Release of lactate dehydrogenase (LDH) was determined as injury index of cardiomyocytes. RESULTS: Adherence of with normal culture group (P < 0.01). The neutrophils to cardiomyocytes with hypoxia/reoxygenation were significantly increased compared lease of LDH by cardiomyocytes was also significantly increased (P < 0.01). There was no significant difference between hypoxia group and normal control (P > 0.05). The adherence of neutrophils to hypoxia/reoxygenation cardiomyocytes was significantly inhibited by anti-ICAM-1 and anti-LFA-1 antibody compared with normal culture group (P < 0.01). While release of LDH markedly decreased (P < 0.01). CONCLUSIONS: Hypoxia/reoxygenation increased the adherence of neutrophils to cardiomyocytes. ICAM-1 and LFA-1 mediated the enhanced adherence of neutrophils to hypoxia/reoxygenation cardiomyocytes. Anti-ICAM-1 and anti-LFA-1 antibody attenuated the cytotoxic effects of neutrophils on hypoxia/reoxygenated cardiomyocytes. These results suggested that the damage of neutrophils on cardiomyocytes is partly mediated by ICAM-1/LFA-1. Anti ICAM-1 and LFA-1 antibody are both beneficial in protecting hypoxia/reoxygenation cardiomyocytes from injury mediated by neutrophils.

Lysophosphatidylcholine activates p38 and p42/44 mitogen-activated protein kinases in monocytic THP-1 cells, but only p38 activation is involved in its stimulated chemotaxis.

Oxidized LDLs (OxLDLs) have been shown to be involved in recruitment of blood monocytes into the arterial subendothelial space, which is the earliest step in atherogenesis, but the underlying molecular mechanisms are poorly understood. The present study demonstrated that lysophosphatidylcholine (LPC), a major phospholipid component of OxLDL, strongly evoked phosphorylation and activation of p38 and p42/44 mitogen-activated protein kinases in monocytic cells. The stimulation of p38 and p42/44 occurred in a dose- and time-dependent manner, reaching the maximal activation at 25 microg/mL LPC within 5 minutes. Interestingly, inhibition of p38 activation by OxLDL or LPC, using its selective inhibitors (SB203580 and SKF86002), completely blocked OxLDL- or LPC-stimulated chemotaxis of THP-1 cells, which was measured in a transwell chemotaxis assay. In contrast, inhibition of p42/44 activation by its potent inhibitor (PD98059) did not block OxLDL- or LPC-stimulated chemotaxis. Moreover, expression of a p38 dominant-negative mutant (p38AF) reduced cell chemotaxis significantly. In addition, activation of p38 by LPC was apparently mediated neither by scavenger receptors nor by tyrosine kinase receptors. It was, however, effectively blocked by pertussis toxin and substantially reduced by phospholipase C inhibitor (U73122) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002). LPC also inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin-sensitive manner, indicating that Gi/Go proteins likely mediated the effects of LPC. Our results suggested that OxLDL/LPC efficiently activated both p38 and p42/44, but only the activation of p38 was functionally associated with OxLDL-/LPC-induced chemotaxis in THP-1 cells.

Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells: mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity.

Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 microg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin-sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs.

Left cervicothoracic sympathetic ganglionectomy with thoracoscope for the treatment of idiopathic long QT syndrome.

A 17-year-old girl had idiopathic long QT syndrome and suffered from recurrent syncopal spells, which were triggered by vigorous physical or emotional stress. She received left cervicothoracic sympathetic ganglionectomy with thoracoscope through an intercostal approach which worked satisfactorily.

Digital color encoding and its application to the moirétechnique.

A moiré pattern color-encoding technique in which a digital image-processing system is used is described. The technique can display color moiré patterns with color sequences that represent the fringe orders. This technique is simple and fast and can be performed automatically. Experimental results demonstrating the feasibility of the technique are reported.

Changes of plasma immuno-reactive beta-endorphin content in patients with congestive heart failure and their clinical significance.

Immunoreactive beta-endorphin (ir-beta-EP) content in plasma was measured by radioimmunoassay in 101 cardiac patients and 30 normal subjects. The results showed that plasma ir-beta-EP levels in patients of Class II (New York Heart Association Classification), Class III and Class IV were significantly higher than those in the normal group and those in patients of Class I (43.14 +/- 2.80, 54.25 +/- 4.47, 79.28 +/- 8.96 ng/L vs 24.23 +/- 2.13, and 24.98 +/- 3.35 ng/L, respectively, P less than 0.01). Plasma concentration of ir-beta-EP in patients complicated with atrial fibrillation was significantly higher than that in patients without atrial fibrillation (56.27 +/- 4.13 vs 44.66 +/- 3.41 ng/L, P less than 0.05). Ir-beta-EP contents in plasma were correlated positively to the cardiothoracic ratios (r = 0.63, P less than 0.001) and PEP/LVET ratios (r = 0.33, P less than 0.01), and were correlated negatively to the left ventricular ejection fraction and axis shortening (r = -0.41 and r = -0.39, P less than 0.001). These results indicated that plasma ir-beta-EP content may serve as a parameter in evaluating cardiac dysfunction.