Identification of novel hub genes associated with gastric cancer using integrated bioinformatics analysis.

BACKGROUND: Gastric cancer (GC) is one of the most common solid malignant tumors worldwide with a high-recurrence-rate. Identifying the molecular signatures and specific biomarkers of GC might provide novel clues for GC prognosis and targeted therapy. METHODS: Gene expression profiles were obtained from the ArrayExpress and Gene Expression Omnibus database. Differentially expressed genes (DEGs) were picked out by R software. The hub genes were screened by cytohubba plugin. Their prognostic values were assessed by Kaplan-Meier survival analyses and the gene expression profiling interactive analysis (GEPIA). Finally, qRT-PCR in GC tissue samples was established to validate these DEGs. RESULTS: Total of 295 DEGs were identified between GC and their corresponding normal adjacent tissue samples in E-MTAB-1440, GSE79973, GSE19826, GSE13911, GSE27342, GSE33335 and GSE56807 datasets, including 117 up-regulated and 178 down-regulated genes. Among them, 7 vital upregulated genes (HMMR, SPP1, FN1, CCNB1, CXCL8, MAD2L1 and CCNA2) were selected. Most of them had a significantly worse prognosis except SPP1. Using qRT-PCR, we validated that their transcriptions in our GC tumor tissue were upregulated except SPP1 and FN1, which correlated with tumor relapse and predicts poorer prognosis in GC patients. CONCLUSIONS: We have identified 5 upregulated DEGs (HMMR, CCNB1, CXCL8, MAD2L1, and CCNA2) in GC patients with poor prognosis using integrated bioinformatical methods, which could be potential biomarkers and therapeutic targets for GC treatment.

Development and validation of a HPLC-MS/MS method for the determination of venlafaxine enantiomers and application to a pharmacokinetic study in healthy Chinese volunteers.

An HPLC-MS/MS method has been developed and validated for the determination of venlafaxine enantiomers in human plasma and applied to a pharmacokinetic study in healthy Chinese volunteers. The method was carried out on a vancomycin chiral column (5 µm, 250 × 4.6 mm) maintained at 25°C. The mobile phase was methanol-water containing 30 mmol/L ammonium acetate, pH 3.3 adjusted with aqueous ammonia (8:92, v/v) at the flow rate 1.0 mL/min. A tandem mass spectrometer with an electrospray interface was operated in the multiple reaction monitoring mode to detect the selected ions pair at m/z 278.0 → 120.8 for venlafaxine enantiomers and m/z 294.8 → 266.7 for estazolanm (internal standard). The method was linear in the concentration range of 0.28-423.0 ng/mL. The lower limit of quantification was 0.28 ng/mL. The intra-and inter-day relative standard deviations were less than 9.7%. The method was successfully applied for the evaluation of pharmacokinetic profiles of venlafaxine enantiomers in 18 healthy volunteers. Validation parameters such as the specificity, linearity, precision, accuracy and stability were evaluated, giving results within the acceptable range. Pharmacokinetic parameters of the venlafaxine enantiomers were measured in the 18 healthy Chinese volunteers who received a single regimen with venlafaxine hydrochloride capsules. The results show that AUC((0-∞)) , C(max) and t(1/2) between S-venlafaxine and R-venlafaxine are significantly different (p < 0.05).

[Expression and prognostic significance of EphA2 and EphrinA-1 in ovarian serous carcinomas].

OBJECTIVE: To investigate the expression and prognostic significance of EphA2 and EphrinA-1 in ovarian serous carcinomas. METHODS: Ninety five tumors from the patients with ovarian serous carcinomas and 2 ovarian cancer cell lines were recruited. The expressions of EphA2 and EphrinA-1 were examined by means of immunohistochemistry. The relationships among protein expression and clinicopathological features, survival of patients were analyzed. The mRNA and protein expressions of EphA2 and EphrinA-1 in ovarian cancer cell lines were measured with semiquantitative polymerase chain reaction and western blotting. RESULTS: The protein expressions of EphA2 and EphrinA-1 in tumor were 92.6% (88/95)and 97.9% (93/95) respectively, while were 40%(8/20) and 30% (6/20) in adjacent ovarian tissue (P = 0.000). EphA2 immunohistochemical staining could be observed in both tumour and vascular endothelial cells, and EphrinA-1 mainly localized in the tumor cells. The expression of EphA2 was significantly associated with the expression of EphrinA-1 (r = 0.98, P = 0.02). There was no significant correlation between the expressions of EphA2/EphrinA-1 and age, FIGO stage, residual tumour size and histological grade. High levels of both EphA2 and EphrinA-1 protein expression were significantly associated with a shorter overall survival in multivariate analysis (P < 0.05). High levels of EphA2 and EphrinA-1 mRNA and protein were detected in OVCAR3 and SKOV3 cell lines. CONCLUSION: There are over-expression of EphA2 and EphrinA-1 in ovarian serous carcinomas and ovarian cancer cell lines. Tumours with higher expression levels of both EphA2 and EphrinA-1 significantly associated with poorer clinical outcome.

Prediction of pandemic risk for animal-origin coronavirus using a deep learning method.

BACKGROUND: Coronaviruses can be isolated from bats, civets, pangolins, birds and other wild animals. As an animal-origin pathogen, coronavirus can cross species barrier and cause pandemic in humans. In this study, a deep learning model for early prediction of pandemic risk was proposed based on the sequences of viral genomes. METHODS: A total of 3257 genomes were downloaded from the Coronavirus Genome Resource Library. We present a deep learning model of cross-species coronavirus infection that combines a bidirectional gated recurrent unit network with a one-dimensional convolution. The genome sequence of animal-origin coronavirus was directly input to extract features and predict pandemic risk. The best performances were explored with the use of pre-trained DNA vector and attention mechanism. The area under the receiver operating characteristic curve (AUROC) and the area under precision-recall curve (AUPR) were used to evaluate the predictive models. RESULTS: The six specific models achieved good performances for the corresponding virus groups (1 for AUROC and 1 for AUPR). The general model with pre-training vector and attention mechanism provided excellent predictions for all virus groups (1 for AUROC and 1 for AUPR) while those without pre-training vector or attention mechanism had obviously reduction of performance (about 5-25%). Re-training experiments showed that the general model has good capabilities of transfer learning (average for six groups: 0.968 for AUROC and 0.942 for AUPR) and should give reasonable prediction for potential pathogen of next pandemic. The artificial negative data with the replacement of the coding region of the spike protein were also predicted correctly (100% accuracy). With the application of the Python programming language, an easy-to-use tool was created to implements our predictor. CONCLUSIONS: Robust deep learning model with pre-training vector and attention mechanism mastered the features from the whole genomes of animal-origin coronaviruses and could predict the risk of cross-species infection for early warning of next pandemic.

UNC0638, a G9a inhibitor, suppresses epithelial­mesenchymal transition­mediated cellular migration and invasion in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is associated with an aggressive clinical history, high risk of recurrence and metastasis, and shorter patient survival due to lack of targeted therapy. In the present study, UNC0638, a chemical G9a inhibitor, was identified to suppress TNBC cell invasion and migration in vitro at a non­cytotoxic concentration. In addition, UNC0638 reduced the size and number of the tumorsphere and decreased anchor­independent colony formation in the two TNBC cell lines. Evaluation of the underlying mechanism revealed that the suppressive effect of UNC0638 is associated with modulation of epithelial­mesenchymal transition through enhancing E­cadherin promoter activities and restoring its expression. Thus, the current data indicates that UNC0638 may be developed as a chemotherapeutic agent to effectively treat metastatic cancers, including TNBC.

Brain-selective mild hypothermia promotes long-term white matter integrity after ischemic stroke in mice.

INTRODUCTION: The neuroprotective effects of hypothermia in acute ischemic stroke are well documented. However, the mechanisms involved in the effects remain to be clearly elucidated and the role of hypothermia on long-term white matter integrity after acute ischemic stroke has yet to be investigated. AIMS: To investigate the role of mild focal hypothermia on long-term white matter (WM) integrity after transient cerebral ischemia. RESULTS: Mild focal hypothermia treatment immediately after ischemic stroke significantly promotes WM integrity 28 days after the occlusion of the middle cerebral artery (MCAO) in mice. Higher integrity of white matter, lower activation of total microglia, less infarct volume, and better neurobehavioral function were detected in hypothermia-treated mice compared to normothermia-treated mice. Furthermore, we found that hypothermia could decrease detrimental M1 phenotype microglia and promote healthy M2 phenotype microglia. In vitro, results also indicated that hypothermia promoted oligodendrocytes differentiation and maturation after oxygen glucose deprivation. CONCLUSION: Hypothermia promotes long-term WM integrity and inhibits neuroinflammation in a mouse model of ischemic brain injury.

[Values of impulse oscillometry for pulmonary function and adhesion molecules in childhood asthma].

OBJECTIVE: Impulse oscillometry (IOS) is a novel technique for the evaluation of pulmonary function. Soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) are definitive indicators for the severity of asthma. This study aimed to explore the relationship of IOS pulmonary function with sICAM-1 and sVCAM-1, and their values in childhood asthma. METHODS: IOS via Master Screen System for pulmonary function was performed in 40 children with acute asthma and 25 healthy children. Twenty-three of 40 children with acute asthma were re-tested for IOS pulmonary function at remission. sICAM-1 and sVCAM-1 levels were measured in 23 children with acute asthma, 20 asthmatic children at remission and 16 healthy children. RESULTS: The parameters of IOS pulmonary function, R5, R20, R5-R20, X5, Fres and Zrs in children with acute asthma were significantly higher than in asthmatic children at remission and in normal controls (q= 2.91-15.61, P < 0.01 or 0.05). There were significant differences in R5, R5-R20, Fres and Zrs between the asthmatic children at remission and normal controls (q= 3.08- 9.19, P < 0.01 or 0.05). sICAM-1 and sVCAM-1 levels in children with acute asthma were significantly higher than in asthmatic children at remission and in normal controls (q= 6.23-26.15, P < 0.01). The asthmatic children at remission had higher levels of sICAM-1 and sVCAM-1 than the normal controls (q=16.86, 12.46, P < 0.01). R5-R20 positively correlated with sICAM-1 and sVCAM-1 in children with acute asthma (r=0.45, 0.57, P <0.05). CONCLUSIONS: IOS for pulmonary function and sICAM-1 and sVCAM-1 may be used to evaluate the severity and therapeutic effects of childhood asthma. A correlation exists between IOS pulmonary function and sICAM-1 and sVCAM-1.

[Effectiveness of Electroacupuncture of Zigong (EX-CA 1), etc. in Treatment of Perimenopausal Syndrome Women].

OBJECTIVE: To evaluate the curative effect of electroacupuncture(EA) of Zigong (EX-CA 1) and Tianshu (ST 25) and acupuncture of Sanyinjiao (SP 6) and Guanyuan (CV 4) for perimenopausal syndrome(PMS). METHODS: Fifty-eight PMS patients were equally and randomized into acupuncture group and medication group. EA (10 Hz/50 Hz, 30 min in duration) was applied at Zigong (EX-CA 1) and Tianshu (ST 25) in combination with manual acupuncture stimulation of Sanyinjiao (SP 6) and Guanyuan (CV 4) by twirling the acupuncture needles with small amplitude and till deqi. The treatment was conducted once every other day, three times a week, 8 weeks altogether. The patients of the medication group were asked to take estradiol valerate (1 mg/time, qd) for 3 weeks, then, to have one week's rest and continuously took estradiol valerate(1 mg/d) and medroxyprogesterone 17-acetate (8 mg/d) for 10 days. The therapeutic effects were assessed by Menopause Rating Scale Questionnaire[MRS, composing of 3 areas:somatic (4 items), psychological(4 items) and urogenital (3 items) domain]. Serum estradiol (E2), follicle stimulating hormone(FSH) and luteotrophic hormone (LH) contents were assayed using ELISA. RESULTS: After the treatment, the scores of the MRS and the contents of serum FSH and LH were significantly decreased in both the acupuncture and medication groups (P<0.01), and serum E2 contents significantly increased in the two groups (P<0.01). No significant differences were found between the two groups in the total effective rates (P>0.05). Eight weeks' follow-up showed that the MRS score of the acupuncture group was significantly lower than that of the medication group (P<0.05). Of the two 29 PMS patients in the medication and acupuncture groups, 3 (10.3%) and 2 (6.9%) cases were under control, 22 (75.9%) and 21 (72.4%) experienced a marked improvement in their symptoms, 3 (10.3%) and 4(13.8%) were effective, 1 (3.4%) and 2(6.9%) invalid, with the effective rates being 96.6% and 93.1%, respectively. Eight weeks' follow-up showed that the long-term effect of the acupuncture therapy was obviously superior to that of the medication (P<0.05) according to MRS score. CONCLUSIONS: Acupuncture therapy is effective in relieving clinical symptoms of PMS women by regulating endocrine hormones, being similar to medication.

Ischemic postconditioning diminishes matrix metalloproteinase 9 expression and attenuates loss of the extracellular matrix proteins in rats following middle cerebral artery occlusion and reperfusion.

AIMS: Ischemic postconditioning (IPostC) has been proved to have neuroprotective effects for cerebral ischemia, but the underlying mechanism remains elusive. This study aimed at validating the neuroprotective effects of IPostC and investigating whether the neuroprotection of IPostC is associated with matrix metalloproteinase 9 (MMP9) and the extracellular matrix proteins, laminin and fibronectin, following cerebral ischemia/reperfusion in rats. METHODS: The rats in middle cerebral artery occlusion (MCAO) group underwent MCAO and reperfusion, and the animals in MCAO + IPostC group were treated by occluding bilateral common carotid arteries for 10 seconds and then reperfusing for 10 seconds for five episodes at the beginning of MCAO. Apoptosis was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of MMP9, laminin, and fibronectin was measured with immunofluorescence and enzyme-linked immunosorbent assay. RESULTS: IPostC reduced brain edema and infarct volume and improved the neurological function. Furthermore, IPostC decreased cell apoptosis compared with the MCAO group. Compared to the MCAO group, IPostC treatment reduced MMP9 expression. Moreover, the results showed that the expression of laminin and fibronectin significantly increased in the MCAO + IPostC group compared to the MCAO group. CONCLUSION: These findings indicated that diminishment of MMP9 expression and the attenuation of degradation of laminin and fibronectin may be involved in the protective mechanisms of postconditioning against cerebral ischemia/reperfusion injury.

AKT/GSK3ß-dependent autophagy contributes to the neuroprotection of limb remote ischemic postconditioning in the transient cerebral ischemic rat model.

BACKGROUND: Limb remote ischemic postconditioning (RIPostC) has been recognized as an applicable strategy in protecting against cerebral ischemic injury. However, the time window for application of limb RIPostC and the mechanisms behind RIPostC are still unclear. AIMS: In this study, we investigated the protective efficacy and the role of autophagy in limb RIPostC using a transient middle cerebral artery occlusion rat model. RESULTS: Limb RIPostC applied in the early phase of reperfusion reduced infarct size and improved neurological function. Autophagy levels in penumbral tissues were elevated in neurons of limb RIPostC rats, with an increase in the phosphorylation of AKT and glycogen synthase kinase 3ß (GSK3ß). Blocking the AKT/GSK3ß pathway via the AKT inhibitor LY294002 prior to limb RIPostC suppressed the RIPostC-induced autophagy and resulted in the activation of caspase-3 in RIPostC rats, suggesting a critical role for AKT/GSK3ß-dependent autophagy in reducing cell death after cerebral ischemia. CONCLUSIONS: These results aid optimization of the time window for RIPostC use and offer novel insight into, and a better understanding of, the protective mechanism of autophagy in limb RIPostC.

[Promoter methylation status of PTEN gene and the effect of induced demethylation in leukemia cell lines].

OBJECTIVE: To explore the mechanism of PTEN gene expression silence in leukemia cells, and the effect of induced PTEN gene expression in leukemia cells. METHODS: Methylation status of PTEN in leukemic cell lines, including HL-60, Nalm-6, NB4, U937, Raji, K562 and KG-1a was assessed by methylation specific PCR (MSP). The cell lines were then treated with different concentrations of methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). After that the changes in PTEN methylation status were detected by MSP, and PTEN mRNA expression level by reverse transcription PCR (RT-PCR). Growth inhibition and apoptosis of HL-60 and Nalm-6 cells induced by 5-Aza-CdR were observed by MTT assay, and Wright and Annexin V staining, respectively. RESULTS: Hypermethylation of PTEN promoter was detected in HL-60, U937, Nalm-6, Raji and KG-1a, while hypomethylation was found in NB4 and K562 by MSP. After 5-Aza-CdR treatment, the hypermethylation status of PTEN promoter in HL-60 and Nalm-6 cells was reversed and their PTEN mRNA expression levels were up regulated in dose dependent manner with the 5-Aza-CdR concentrations, and the cell apoptosis was induced. CONCLUSION: Hypermethylation in the promoter region is one of major mechanisms responsible for transcriptional suppression of PTEN. Methyltransferase inhibitor could induce the expression of PTEN gene and lead to the leukemia cells apoptosis.

[The expression of midkine in acute leukemia and its significance].

OBJECTIVE: To analyze the expression of midkine (MK) gene in acute leukemia patients, and explore the relationship between the gene and leukemia. METHODS: The MK gene expression levels were detected by real-time quantitative RT-PCR (RQ-RT-PCR) in bone marrow (BM) of 181 acute leukemia (AL) patients and 31 normal controls. RESULTS: MK gene was expressed in all AL patients, normal controls and AL patients in complete remission (CR). Compared with that in control group and CR group, MK gene expression was significantly increased in AL patients (P < 0.01 and P < 0.05, respectively). No statistical difference was found between CR group and control group. The expression of MK showed a notable increase in all B-ALL subtypes (including pro-B-ALL, common-B-ALL and pre-B-ALL) as well as in adult and childhood B-ALL patients (P < 0.01). Moreover, the gene expression in B-ALL was also significantly higher than that in TALL, HAL and FAB subtypes of AML (P < 0.01). In addition, M2 patients showed significantly increased in MK expression compared with that in normal controls (P < 0.01) and in other FAB subtypes of AML (P < 0.05). Median MK expression level in M3 patients was also significantly higher than that in normal controls (P < 0.05), but there was no statistical difference between M3 and other AL subtypes excepting for M2 and B-ALL. MK expression in CD34 positive patients was significantly higher than that in CD34 negative ones (P < 0.01) and within M2 patients, MK expression was higher in patients with t (8 ;21) than in those without the translocation (P < 0.05). CONCLUSION: MK gene expression is increased with different levels in B-ALL, M2 and M3 patients, which provides novel insights into the leukemogenesis of acute leukemia.

[Effects of AML1-ETO on transcription activity of p21WAF1/CIP1 gene promoter].

OBJECTIVE: To observe the effects of AML1-ETO fusion gene on the transcription activity of p21WAF1/CIP1 gene. And to explore the enhancement of leukemia pathogenesis of AML1-ETO. METHODS: The luciferase reporter plasmids of p21WAF1/CIP1 gene promoter were constructed, and co-transfected into CV-1 cells with AML1-ETO, AML1b and AML1a expression plasmids. The trans-activity of p21WAF1/CIP1 gene promoter was assayed by luminometer. RESULTS: AML1-ETO exhibited a distinct inhibition activity of p21WAF1/CIP1 gene promoter with a sequence-specificity and dosage-dependent manner. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (19 +/- 4)% compared to control group, when 1000 ng pCMV5-AML1-ETO plasmid was used. AML1b and AMLla showed less inhibition activity. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (61 +/- 16)% and (59 +/- 16)% compared to control group, respectively, when 1000 ng plasmid was used. CONCLUSION: AML1-ETO exhibits more inhibition activity of p21WAF1/CIP1 gene promoter than AML1b and AMLla, results from recruiting transcription co-repression complex efficiently by ETO. Based on previous researches, the effects of exogenous AML1-ETO on p21WAF1/CIP1 gene promote may be dependent on the type of cell lines.

[Phenol butyrate inhibits cell cycle of leukemia cell lines through up-regulation of p21WAF1/CIP1 gene].

OBJECTIVE: To elucidate effects of histone deacetylase inhibitors on cell cycle of leukemia cell lines and investigate its molecular mechanisms. METHODS: Kasumi-1, U937 and NB4 cell lines were exposed to a histone deacetylase inhibitor, phenyl butyrate (PB), for 24, 48 and 72 hrs. Cells were harvested for cell cycle analysis by flow cytometry. Gene expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, was determined by semi-quantitative reverse transcriptase polymerase chain reaction (semi-quantitative RT-PCR). Promoter activity of p21WAF1/CIP1 was determined by luciferase-reporter assay in 293T cell line. RESULTS: PB inhibited cell cycle of Kasumi-1, U937 and NB4 cell lines, showing G(0)/G(1) phase arrest and S-phase fraction reduction with a dose and time dependent manner. After Kasumi-1, U937 or NB4 cells exposed to 3 mmol/L PB for 72 hrs, G(0)/G(1)-phase fraction was increased by 42.03%, 44.36% and 26.82%, and S-phase fraction was decreased by 31.86%, 38.9% and 26.77%, respectively. After Kasumi-1, U937 and NB4 cell lines exposed to PB, the expression of p21WAF1/CIP1 gene was increased by (2.06 +/- 0.27), (2.78 +/- 0.40) and (1.78 +/- 0.20) times at its maximum, respectively. PB could stimulate p21WAF1/CIP1 promoter activity (by luciferase-reporter assay) and the effect was dose dependent. The promoter activity was increased by 5.74 times after the cells exposed to 3 mmol/L PB for 48 hrs. PB stimulating p21WAF1/CIP1 promoter activity was mainly mediated by a 101 base pairs fragment upstream of transcription start site. CONCLUSION: PB could inhibit cell cycle of leukemia cell lines. The effects were mainly through up-regulation of p21WAF1/CIP1 expression.

[The heat shock protein 90 inhibitor induces apoptosis and differentiation of Kasumi-1 and its mechanisms].

OBJECTIVE: To explore the effect of 17-allylamide-17-demethoxygeldanamycin (17AAG), a heat shock protein 90 (HSP90) inhibitor, on the growth, differentiation and apoptosis of leukemic Kasumi-1 cells. METHODS: Kasumi-1 cells were treated with 17AAG at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry. KIT protein was analysed by Western blot and c-kit mRNA by RT-PCR. RESULTS: 17AAG treatment caused a dose-dependent inhibition of the cell proliferation with the IC(50) of 0.62 micromol/L. A dose-dependent increase in early apoptosis occurred at 24 hours treatment and in late apoptosis at 48 hours treatment. 17AAG induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD11b and CD15, a progressive decline in S-phase cell fraction and an increase in G(0)/G(1) cells. When Kasumi-1 cells were incubated with 1 micromol/L of 17AAG, KIT protein began to decrease at 2 hours and KIT protein could hardly be detected at 20 hours, but c-kit mRNA was not decreased. CONCLUSION: 17AAG treatment of Kasumi-1 cells could lower KIT protein expression, inhibit cell proliferation, induce cell partial differentiation, apoptosis and accumulation in G(0)/G(1) phase.