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Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a major advance in treating NSCLC with EGFR-activating mutations. However, acquired resistance, due partially to secondary mutations limits their use. Here we report that NSCLC cells with acquired resistance to gefitinib or osimertinib (AZD9291) exhibit EMT features, with a decrease in E-cadherin, and increases in vimentin and stemness, without possessing any EGFR secondary mutations. Knockdown of E-cadherin in parental cells increased gefitinib resistance and stemness, while knockdown of vimentin in resistant cells resulted in opposite effects. Src activation and Hakai upregulation were found in gefitinib-resistant cells. Knockdown of Hakai elevated E-cadherin expression, attenuated stemness, and resensitized the cells to gefitinib. Clinical cancer specimens with acquired gefitinib resistance also showed a decrease in E-cadherin and an increase in Hakai expression. The dual HDAC and HMGR inhibitor JMF3086 inhibited the Src/Hakai and Hakai/E-cadherin interaction to reverse E-cadherin expression, and attenuated vimentin and stemness to restore gefitinib sensitivity. The EMT features of AZD9291-resistant H1975 cells were related to the upregulation of Zeb1. Both gefitinib and AZD9291 sensitivity was restored by JMF3086 through reversing EMT. Our study not only revealed a common mechanism of EMT in both gefitinib and AZD9291 resistance beyond EGFR mutations per se, but also provides a new strategy to overcome it.

Objective: : Coagulation factor VII (FVII) triggers the extrinsic pathway of blood coagulation. In our previous study, we showed that FVII plays an important role in tumorigenesis of hepatocellular carcinoma (HCC). However, the role of FVII polymorphism in HCC is still unknown. The present study aimed to investigate the relationship between HCC carcinogenesis and single nucleotide polymorphism of FVII., Methods: : Thirty-seven HCC patients and 30 healthy donors were recruited in this study. Four common FVII gene polymorphisms - a decanucleotide insertion at position -323 (-323ins10-bp), a G to T substitution at position -401 (-401G/T), a G to A substitution at position -402 (-402G/A), and a T to C substitution at position -122 (-122T/C) - were analyzed by sequencing or commercialized assays using genomic DNA isolated from blood samples. Clinicopathological parameters between control and HCC subjects were compared according to the specific genotypes., Results: : The most common nucleotide variation was -402G/A. However, no statistically significant difference was observed between healthy controls and HCC subjects for all four polymorphisms in terms of genotype distribution and allele frequencies, indicating that these polymorphisms may not affect HCC tumorigenesis. Furthermore, no association was found between -402G/A polymorphisms and tumor stage, recurrence, and overall survival., Conclusions: : Our results indicate that FVII polymorphisms may not be a key factor that clinically impact tumorigenesis and outcomes of HCC, although further investigations should be conducted to confirm our findings.

Objective: To compare clinical characteristics and therapeutic outcomes between HIV and non-HIV patients with Cryptococcus neoformans meningitis (CNM)., Methods: A total of 73 patients with CNM (30 patients without HIV and 43 with HIV) were admitted from January 2012 to January 2017. The clinical manifestations,biochemical and microbiological characteristics of cerebrospinal fluid (CSF) were collected and analyzed., Results: The patients in the two group displayed non-specific symptoms such as headache,fever,nausea and vomiting. Non-HIV CNM patients had more serious inflammatory reaction with higher karyocytes and protein level ( P =0.000, P =0.041,respectively),while had lowere positive rate of primary ink staining in cerebrospinal fluid (70.0% vs. 93.0%, P =0.009),higher misdiagnosis rate (43.3% vs. 14.0%, P =0.005),longer hospitalization duration [(112.27±105.42) d vs. (52.64±39.17) d, P =0.021],higher adverse reactions rate of antifungal treatment was (80.0% vs. 30.2%, P =0.000). However,in HIV CNM patients,40 (93.0%) patients did not receive antiviral therapy before and were diagnosed as AIDS for the first time; the therapeutic effect in this group was very poor with higher mortality (30.2% vs. 13.4%, P =0.000)., Conclusion: Immunity status should be considered in the diagnosis and treatment of CNM,since it is difficult to diagnose with long treatment period and poor prognosis., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Background/aim: This study aimed to investigate the potential regulatory role of prokineticin 2 (PK2) in modulation of the number and function of Kupffer cells (KCs) during the progression of liver fibrosis in patients with hepatitis B virus (HBV). Materials and methods: We obtained liver tissue sections from 200 patients with HBV undergoing surgical resection in our hospital between January 2013 and July 2016. Of these 200 tissue sections, 150 were fibrosis tissues and 50 were hepatocellular carcinoma tissues. Immunohistochemical evaluations were performed to assess the expression levels of CD68 and PK2 in the sections. The clinical parameters of these 200 patients were also analyzed. Results: As a potential cytokine, PK2 was commonly expressed in KCs. In addition, a close correlation between PK2 and the number of KCs during the progression of liver fibrosis in patients with HBV was found in this study. Conclusion: PK2 is expressed in KCs and participates in the progression of liver fibrosis after HBV infection. As a potential cytokine, PK2 modulates the number of KCs during fibrosis. Thus, PK2 most likely adjusts the number of M1 cells to modulate the role of KCs in the progression of liver fibrosis after HBV infection.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide. Treatment outcomes remain poor mainly due to lack of good diagnostic/prognostic markers and limited therapeutic strategies. We previously characterized aberrant activation of the TF/FVII/PAR2 pathway, which subsequently results in decreased autophagy, as a crucial event in malignant progression of HCC. Here, we identified miR-135a as a highly upregulated miRNA in HCC in response to TF/FVII/PAR2 activation. Analyzing 103 HCC patient specimens, we confirmed that miR-135a was frequently elevated in HCC tissues with higher FVII expression compared to adjacent non-cancerous counterparts. Increased miR-135a levels in HCC were also associated with tumor staging, recurrence, microvascular invasion, and decreased disease-free survival. We subsequently identified Atg14, a key component that regulates the formation of autophagosome as a direct target of miR-135a. Ectopic expression of miR-135a suppressed Atg14 levels and inhibited the autophagic processes. Our results indicate strong positive correlations between miR-135a levels and malignant behaviors in HCC patients and also suggest novel functions of miR-135a in regulation of autophagy, which could be useful as a potential target for prognostic and therapeutic uses., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: There are increasing concerns regarding the adverse effects associated with control ovarian hyperstimulation (COH) in both endometrial and uterine environments. With the "segmentation treatment policy" of assisted reproductive techniques (ART), endometrial problems may be obviated through embryo cryopreservation. However, it remains unclear if the "freeze-all policy" offers a better outcome when compared with fresh embryo transfer (ET). To clarify this, we compared the cumulative live birth rates (CLBRs) between these two patient populations., Materials and Methods: This is a retrospective study on 853 patients undergoing ovarian stimulation and ART (including IVF/ICSI) during the period from January 2012 to June 2014 in Taichung Veterans General Hospital, Taiwan, ROC. We followed up with these patients through to November 2016. Patients whose embryos were not completely transferred back were excluded. The study group ('freeze-all') included 84 patients whose cycles were performed initially without fresh ET, but were later given frozen-thawed ET. The control group ('fresh ET') had 625 patients whose cycles were performed with fresh ET, followed by frozen-thawed ET. Basic parameters and CLBRs were statistically compared between these two groups., Results: The CLBRs in the study group were significantly higher than those in the control group (64.3% vs. 45.8%, p = 0.001). Subgroup analysis revealed that when the number of oocyte pick up (OPU) is between 4 and 15, the CLBRs in the study group were significantly better (58.3% vs. 40.9%, p = 0.042). For those with OPU <4 or OPU >15 the CLBRs were similar in these two groups (OPU < 4: study vs. control 23.1% vs. 18.8% respectively, p = 0.713; OPU>15: study vs. control 85.7% vs. 80.8% respectively, p = 0.625) CONCLUSION: The Freeze-all policy improved the ART outcome for normal responders., (Copyright © 2017. Published by Elsevier B.V.)

Aim: To investigate the targeted inhibition of proliferation and migration of SW620 human colon cancer cells by upregulating miRNA-145 (miR-145)., Methods: Forty-five samples of colon cancer tissues and 45 normal control samples were obtained from the biological database of the First Affiliated Hospital of Liaoning Medical University. We performed quantitative analysis of miR-145 and N-ras expression in tissues; reverse transcriptase polymerase chain reaction analysis of miR-145 expression in SW620 colon cancer cells and normal colonic epithelial cells; construction of miR-145 lentiviral vector and determination of miR-145 expression in SW620 cells transduced with miR-145 vector; analysis of the effect of miR-145 overexpression on SW620 cell proliferation; analysis of the effect of miR-145 overexpression on SW620 cell migration using a wound healing assay; and analysis of the effect of miR-145 on N-ras expression using Western blotting., Results: miR-145 expression was significantly downregulated in colon cancer tissues, with its expression in normal colonic tissues being 4-5-fold higher (two sample t test, P < 0.05), whereas N-ras expression showed the opposite trend. miR-145 expression in SW620 cells was downregulated, which was significantly lower compared to that in colonic epithelial cells (two sample t test, P < 0.05). miR-145 vector and control were successfully packaged; expression of miR-145 in SW620 cells transduced with miR-145 was 8.2-fold of that in control cells (two sample t test, P < 0.05). The proliferation of miR-145-transduced SW620 cells was significantly decreased compared to control cells (two sample t test, P < 0.05). At 48 h in the wound healing experiment, the migration indexes and controls were (97.27% ± 9.25%) and (70.22% ± 6.53%), respectively (two sample t test, P < 0.05). N-ras expression in miR-145-tranduced SW620 cells was significantly lower than others (one-way analysis of variance, P < 0.05)., Conclusion: miR-145 is important in inhibiting colon cancer cell proliferation and migration. This is a good foundation for development of colon cancer therapy by targeting tumor suppressor miR-145.

Objective: To analyze the correlation between serological indices and liver histological pathology in the patients with hepatitis B virus ( HBV) infection., Methods: This study enrolled 301 patients with HBV infection. All of them received liver biopsy, serum biochemical examination, including alanineamino transferase (ALT), aspart ateaminotransferase (AST), albumin (ALB), globulin (GLB ), HBV DNA test and HBV genotype detection., Results: ALT, AST and AST/ALT in G2/G(3+4) group were significantly higher than those in group G0/G1 (P < 0.05), and all showed positive correlation with liver inflammation (r = 0.487, 0.648, 0.509, P < 0.05). GLB in S2/S(3+4) group was significantly higher than that in group S0/S1 (P < 0.05), which had positive correlation with liver fibrosis (r = 0.674, P < 0.05). ALB/GLB (A/G) in S2/S(3+4) group was significantly lower than that in group S0/S1 (P < 0.05), it had negative correlation with liver fibrosis(r = -0.500, P < 0.05). The inflammation and fibrosis level in patients with C genotype was higher than that of B genotype (χ2 = 11.460, 12.729, P < 0.05)., Conclusion: ALT, AST and AST/ALT show better diagnostic value in liver inflammation. GLB and A/G show better diagnostic value in liver fibrosis. The progress of this disease is relatively faster in the patients with C genotype HBV infection.

Aim: To study the potential prognostic role of microRNA-382 (miR-382) in esophageal squamous cell carcinoma (ESCC)., Methods: Forty six patients were divided into 2 groups according to postoperative survival time: the poor outcome group (28 patients), who showed early metastasis but no recurrence, and died within 1 year after surgery, 12 patients of the group received postoperative chemotherapy treatment that was given after early metastasis happening; the good outcome group (18 patients), who had no clinical metastasis and recurrence, and survived 5 years or more after surgery, all patients did not receive any postoperative treatment. Total RNA was extracted from the patients' formalin-fixed and paraffin-embedded esophageal cancer tissues. miR-382 level was evaluated using high-throughput real-time quantitative polymerase chain reaction analysis. The correlation between miR-382 level and clinicopathologic features was analyzed through COX regression model, and Kaplan-Meier analysis was used to analyze the relationship between miR-382 level and patient survival time., Results: miR-382 was differentially expressed in the two groups. Overall the average miR-382 level in the ESCC patients with good outcome was 9.8 ± 3.8, while miR-382 level in the ESCC patients with poor outcome was 3.0 ± 0.8. The differences of miR-382 levels between two groups were significant (P < 0.05). Kaplan-Meier analysis results showed that miR-382 expression level generally had a significant reverse-correlation with ESCC patient survival time (P < 0.001), in which the patients with higher expressions of miR-382 had a longer survival time either among individuals with the same tumor stage or among the overall patients., Conclusion: miR-382 levels are reverse-correlated with ESCC poor outcomes, suggesting that miR-382 could be a potential predictive biomarker for both prognosis and treatment of ESCC.

Objective: To explore the predictive value of serum hepatitis B surface antigen (HBsAg) titer and transient elastography in screening for insignificant fibrosis in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients., Methods: We conducted a cross-sectional study of eligible patients treated from March 2012 to May 2013 at the West China Hospital of Sichuan University. Eligible patients underwent liver transient elastography and liver biopsy. We assessed the serum HBsAg level, serum hepatitis B virus (HBV) deoxyribonucleic acid (DNA) level, HBV genotypes, liver stiffness measurement (LSM) values by transient elastography, and histological fibrosis staging by METAVIR classification., Results: A total of 129 consecutive patients were recruited. The LSM value (P<0.001, odds ratio 14.67, 95% CI 0.158-0.551) and log10HBsAg (P=0.045, odds ratio 4.03, 95% CI 0.136-0.976) correlated with a liver fibrosis score

The aim of the study was to investigate the feasibility and value of clinical application of fine-needle aspiration histological biopsy via ultrasound-guided thyroid nodule and enlarged cervical lymph node fine-needle aspiration histological biopsy. Fine-needle aspiration cytological and histological biopsies and surgical treatments were performed on 982 patients with thyroid nodule and 1435 patients with enlarged cervical lymph nodes. A comparative study of the histological and cytological examination results and post-surgical etiology results was subsequently conducted. Among the 982 thyroid nodule patients, the acquisition rates were 89.8% (882/982) for fine-needle aspiration histological biopsy and 96.2% (945/982) for cytological biopsy, while among the 1435 patients with enlarged cervical lymph nodes, the acquisition rate for fine-needle aspiration cytological biopsy was slightly higher than that for histological biopsy, with values of 95.7% (1374/1435) and 91.4% (1312/1435), respectively. For the thyroid nodule patients, when the acquired histological and cytological biopsy results were compared with the post-surgical etiology results, the sensitivity, specificity, and accuracy of the histological results were 98.5%, 100%, and 98.9%, respectively, whereas those of the cytological results were 86.8%, 82.9%, and 85.6%, respectively; the differences between the 2 biopsy methods were statistically significant (P < 0.05). For the patients with enlarged cervical lymph nodes, when the acquired histological and cytological biopsy results were compared with the post-surgical etiology results, the sensitivity, specificity, and accuracy of the histological results were 96.3%, 99.8%, and 97.6%, respectively, whereas the those of the cytological results were 76.8%, 92.1%, and 82.2%, respectively; again, the differences between the 2 methods were statistically significant (P < 0.05). In conclusion, Fine-needle aspiration histological biopsy is a reliable and highly accurate examination method. It is simple and feasible, thus facilitating the discrimination of malignant and benign thyroid nodules and enlarged cervical lymph nodes and playing an important role in the establishment of reasonable clinical therapeutic regimens.

Objective: To determine the prevalence of type 2 DM and impaired glucose regulation (IGR) in Chengdu populations and to identify dietary risk factors associated with DM and IGR., Methods: Two communities in Chengdu were selected for this study. Fasting blood-glucose (FBG) and 2-hour post-meal blood glucose (2 hGlu) tests were performed in the community residents. The participants were asked to complete a questionair recording their daily food intaking. The total calorie of food, percentage of different kinds of food, and intake of electrolyte, vietamine and micro minerals were calculated and compared between those with and without type 2 DM or IGR., Results: Of the study participants, 18.59% had type 2 DM and 24.22% had IGR. Those with DM had higher levels of intake of calorie,fat,protein and sodium, and lower levels of intake of cellulose, carbohydrates, Iron, zinc, selenium,manganese and vietamine C and E compared with those without DM/IGR (P < 0.05). The participants with IGR had higher levels of intake of calorie and sodium and lower levels of intake of cellulose, zinc, selenium, manganese and vitamine C and E compared with those without DM/IGR (P < 0.05). Percentage of dietary fat was identified as an independent risk factor of type 2 DM (OR = 1.609); whearaus Vitamine E was identified as a protective factor of type 2 DM (OR = 0.733) and IGR (OR = 0.990)., Conclusion: Chengdu has a higher than national average prevalence of type 2 DM and IGR. The high percentage of dietary fat and low levels of Vitamine E are major risk factors of type 2 DM and IGR.

To evaluate the therapeutic efficacy of antiviral combination therapy with pegylated-interferon alpha-2a plus ribavirin (RBV) in patients with autoantibody-positive chronic hepatitis C (CHC) and to investigate the impact of the presence of autoantibodies on the treatment outcome. Eighty-six consecutive CHC patients who underwent a 48-week treatment regimen composed of Peg-IFNa-2a (135 or 180 mug/wk) plus weight-based RBV ( less than or equal to 65 kg, 800 mg/d; 65 to 75 kg, 1000 mg/d; more than or equal to75 kg, 1200 mg/d ). Prior to treatment (baseline) and at end of treatment (EOT; week 48), levels of antinuclear antibody (ANA), anti-smooth muscle antibody (SMA), anti liver/kidney microsomal antibody type 1 (LKM1), anti-La (SSB), and anti liver cytosolic-1 (LC-1) were detected by indirect immunofluorescence. At baseline, during treatment (weeks 4, 12, 24, and 36), EOT, and 24 weeks after EOT, levels of HCV RNA were assessed by real-time quantitative PCR. Rapid virological response (RVR) was defined as HCV RNA less than 10(3) copy/ml at week 4. Sustained virologic response (SVR) was defined as HCV RNA load below the lower limit of detection at 24 weeks after EOT. Correlation between autoantibodies and treatment-induced reduced HCV RNA load was assessed by univariate analysis of variance or chi-squared tests. Autoantibodies were detected in 24 patients, which included 14 ANA-positive patients, five SMA-positive patients, three LKM1-positive patients, one patient with double-positivity for ANA and SSB, and one patient with double-positivity for ANA and LC-1. The autoantibody-positive patients and autoantibody-negative patients showed similar rates of RVR (70.8% vs. 72.5%, P more than 0.05) and SVR (81.4% vs. 82.2%, P more than 0.05). Antiviral therapy with Peg-IFNa-2a RBV can effectively reduce the HCV RNA load in autoantibody-positive CHC patients; however, the presence of autoantibodies may not be an independent predictor of therapy outcome.

Objective: To analyze whether or not the accuracy of liver stiffness measurement (LSM) with transient elastography (FibroScan) for the diagnosis of liver fibrosis influenced by serum alanine aminotransferase (ALT) levels in patients with chronic hepatitis B., Methods: 213 consecutive CHB patients who underwent liver biopsy and LSM were enrolled and divided into three groups by the criteria of ALT < 1 x ULN, 1 x ULN ≤ ALT < 2 x ULN and ALT ≥ 2 x ULN. The areas under the receiver operating curve(AUC) were analyzed and the accuracy of FibroScan for the diagnosis of liver fibrosis were detected in the three groups., Results: Significant correlation existed between the stages of liver fibrosis and LSM (rs = 0.773, 0.889 and 0.412, P < 0.05). AUCs of LSM in all patients for significant fibrosis (F ≥ 2, F0-1 vs F2-4) and cirrhosis (F = 4, F0-3 vs F4) were 0.916 and 0.971 respectively. The accuracy of diagnosis for significant fibrosis and cirrhosis were 84.0% and 93.4% respectively. AUCs of LSM in ALT < 1 x ULN, 1 x ULN ≤ ALT < 2 x ULN and ALT ≥ 2 x ULN groups for significant fibrosis were 0.939, 0.967 and 0.687 respectively. The sensitivity of the three groups was 90.0%, 89.7% and 47.8% respectively. The accuracies of the three groups was 90.5%, 93.9% and 68.4% respectively. The AUC, sensitivity and accuracy of ALT ≥ 2 x ULN group for significant fibrosis were significantly lower than the other two groups. AUCs of LSM in three groups for cirrhosis were 0.970, 0.985 and 0.952 respectively. The sensitivities of the three groups were 93.8%, 100% and 100% respectively. The accuracies of the three groups were 88.9%, 95.9% and 92.1% respectively. The AUCs, sensitivity and accuracy for cirrhosis of the three groups didn't change with elevated ALT., Conclusions: Transient elastography (FibroScan) is a reasonable noninvasive tool to diagnose significant fibrosis, especially liver cirrhosis in CHB patients. The accuracy of FibroScan for diagnosis of liver cirrhosis may not be influenced by elevated ALT. While in ALT ≥ 2 x ULN group, the accuracy of FibroScan for diagnosis of significant fibrosis was significantly lower as compared to the ALT ≤ 2 x ULN groups.

Objective: To explore the value of ultrasono-portography using SonoVue in selective portal vein embolization (SPVE)., Methods: Twenty-eight patients with malignant liver tumors underwent percutaneous ultrasound-guided SPVE. The procedure was performed under color Doppler ultrasound guidance in 11 cases (conventional group) and under guidance with ultrasono-portography using SonoVue in 17 cases (contrast group). Contrast-enhanced CT was performed 2-4 weeks after SPVE to evaluate the effect of embolization., Results: The procedure of SPVE was aborted in 3 cases in which ultrasono-portography showed contraindications. Postoperative contrast-enhanced CT showed ectopic embolization in 2 cases in the conventional group, and none of the cases in the contrast group showed ectopic embolization., Conclusion: Ultrasono-portography using SonoVue can provide important assistance for SPVE.

IN VITRO plant regeneration of Gentiana macrophylla Pall. and determination of gentiopicroside content during somatic embryogenesis are described in the present work. The highest percentage of embryogenic callus formation was observed in Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L 6-benzylaminopurine (BA). Calli were subcultured on MS medium containing 1.0 mg/L 2,4-D, 1.0 mg/L BA and 500 mg/L lactalbumin hydrolysate (LH) at intervals of 25 days. A higher frequency of somatic embryo maturation was achieved on MS medium containing B5 vitamins (MB) supplemented with different concentrations of 1-naphthaleneacetic acid (NAA) and BA than with a combination of NAA and kinetin (KT). Addition of AgNO(3) improved maturation of somatic embryos while thidiazuron (TDZ) promoted vitrification. The gentiopicroside contents of embryogenic calli and globular-, heart-, torpedo-, and cotyledon-shaped embryoids were determined by high-performance liquid chromatography (HPLC). Gentiopicroside was not detectable in embryogenic calli, but in all types of somatic embryos. The highest gentiopicroside content was observed in cotyledon-shaped embryoids, reaching more than 12 mg/g dry weight., (Georg Thieme Verlag KG Stuttgart. New York.)

Qinjiao (Gentiana macrophylla Pall.) is a perennial herbage native to northwestern China. It has been taken as a kind of Chinese herbs for more than one thousand years. The major secondary metabolite named as gentiopicroside accounts for 8% of the dry weight of roots. It has been used for medical purpose in the treatment with rheumatism, osteoarthritis, inflammatory or ulceration. Conventionally, Qinjiao is propagated by seeds, but the seeds should be planted in one year after harvesting and the germination rate is extremely low. These confine its spread by seeds after year-round storage. Therefore, plant regeneration from somatic cells will be an ideal way for its propagation. Plant regeneration from somatic cell can be divided into two ways: somatic embryogenesis and organogenesis. Similar to zygotic embryo, somatic embryo has a bipolar structure with both shoot and root poles. Somatic embryogenesis also goes through the same key stages as globular-, heart-, torpedo- and cotyledon-stages Somatic embryogenesis was first observed in carrot callus cells more than 45 years ago. Since then, plant regeneration via somatic embryogenesis has been studied in many important species. Though the plant regeneration of Qinjiao has been previously studied, there is no investigation of somatic embryogenesis reported in this plant. Therefore, the aim of our experiment is to confirm the somatic embryogenesis pathway of plant regeneration in Qinjiao.

Objective: To determine whether U(251) cells are permissive for human cytomegalovirus (HCMV), and to investigate the characteristics of temporal expression of proteins IE1 and pp65., Methods: U(251) cells were infected with HCMV, and then the cells were observed under the transmission electronic microscope, and the viral nucleic acid was detected by PCR, and the expression levels of IE1 and pp65 were analyzed by immunohistochemical assay with anti-IE1 monoclonal antibody and anti-pp65 monoclonal antibody at various time spost infection., Results: Morphological changes of the infected cells appeared under the transmission electron microscope. The viral nucleic acid was detected successfully by PCR. The expression of IE1 was detected firstly at 4h post infection, and reached a peak within 14h, and then decreased. The incoming pp65 was detected at 1h, the low expression levels of pp65 were detected firstly at 4h, and they could remain relatively constant through 96 h, but the maximum expression occurred at 120 h., Conclusion: Human glioma U(251) cells are permissive for HCMV, the temporal cascade of HCMV gene expression can be observed in the infected U(251) cells, but it is delayed obviously in the human fibroblast.

In HCl-NaAc buffer solution, the hydroxy free radical from the Fenton reaction is captured by excess KI and releases I3-. The I3- combines with Rhodamine B (RhB, lamdamax=554 nm), Rhodamine 6G(Rh6G, lamdamax=526 nm), Rhodamine S (RhS, lamdamax=526 nm), and butyl Rhodamine B(b -RhB, A,lamdamax56 nm) to form association particles, so the absorbance at max wavelength decreases. The concentration of hydroxy free radical (calculated by the concentration of hydrogen peroxide) is proportional to the decreased absorbance of the systems of RhB, Rh6G, RhS, b-RhB in the range of 0.136-0.680 microg x L(-1), 0.34-0.680 microg x mL(-1), 0.034-0.680 microg x mL(-1), 0.034-0.680 microg c mL(-1), espectively. Based on this fact, a new method for the determination of scavenging percentage of hydroxy free radical with antioxidant was developed. The resistance to oxidation of four substances and six kinds of tea extract were measured with satisfactory results.

Objective: To study the changes of p38MAPK expressions, the frequency of apoptosis and the distribution of cell cycle of hunan Glioma U251 cells after HCMV infection., Methods: The expression of total p38 (both phosphorylated and nonphosphorylated p38) and phosphorylated p38 in U251 cells were detected by Western blotting at 15 min, 30 min, 1 h, 6 h, 10 h, 16 h, 24 h, 36 h and 48 h after HCMV infection. The apoptosis percentage and the cell cycle distribution of U251 cells at 2 d, 5 d and 7 d after HCMV infection were detected by flow cytometry (FCM)., Results: The results of Western blotting demonstrated that a strong increase in phosphorylated p38 was detected from 6 h to 10 h after HCMV infection, with mean gray scales 186.33 +/- 7.51 (t = 5.37, P < 0.01) and 188.00 +/- 7.02 (t = 5.26, P < 0.01 for all) at 6 h and 10 h, respectively, and p38 phosphorylation decreased to the basic level at 16 h after HCMV infection. But the overall levels of p38 protein were not significantly altered during the course of infection. FCM analysis showed that HCMV could significantly increase the apoptotic rates of U251 cells compared with controls (t = 10.84, P < 0.01), and the apoptotic percentages of the cells reached to peak [(10.18 +/- 1.24)%] at 5 d after HCMV infection. The data of FCM showed that HCMV could decrease the number of U251 cells in G1 phase and arrest the cells in S and G2 phase. The numbers of G1 phase U251 cells were significantly lowered to (56.50 +/- 2.57)% (t = 26.45, P < 0.01), (62.33 +/- 2.64)% (t = 21.20, P < 0.01) and (67.45 +/- 4.44)% (t = 10.61, P < 0.01), respectively at 2 d, 5 d and 7 d after infection., Conclusion: HCMV could activate p38MAPK pathway and trigger apoptosis and interfere cell cycle in U251 cells.

Specific primer sequences were designed on the basis of converse motifs of cloned resistance genes and subsequently used as PCR primers to amplify the resistance gene analog (RGA) in wheat (Triticum aestivum L.) Xinong 88 which possesses rust disease resistance. The amplified products of WRGA1, WRGA2 and WRGA14 were cloned and sequenced. The wheat RGAs contain some conserved motifs such as Kinase-2a, Kinase-3a and HD presenting in known NBS-LRR resistance genes from other plants and share between 46.0%-9.9% amino acid sequence identity with them. The amino acid sequence identity among three RGAs is 80.7%-56.8%. The WRGA1 expression was induced and detected by Northern blotting, which is positively regulated by salicylic acid.

Objective: To compare clinical therapeutic effects of phased integral acupuncture and routine acupuncture on patients with cerebral infarction., Methods: One hundred and thirteen cases were randomly divided into a treatment group (n = 63) treated by phased integral acupuncture and a control group (n = 50) treated by routine acupuncture. Their clinical therapeutic effects were compared after treatment., Results: The treatment group in the decreases of both the diagnostic score for TCM diseases of stroke and the score for neurological function defect, and the therapeutic effect was superior to the control group with significant differences (all P < 0.05)., Conclusion: The phased integral acupuncture is an effective therapy with a therapeutic effect better than that of the routine acupuncture for treatment of hemiplegia due to cerebral infarction.

Objective: To subclone and express the gene encoding Schistosoma japonicum ferritin (SjFer) and study its immune protection against challenge infection in mice vaccinated intranasally., Methods: The SjFer gene was amplified by PCR, and subcloned into the N-terminal of intein 2 of the pTWIN1 vector. The positive recombinant was screened by PCR, restriction enzyme digestion and sequence analysis. The positive recombinant was transformed into E. coli ER2566. The soluble recombinant fusion protein (rSjFer-intein 2) was expressed in E. coli by induction of low IPTG concentration under low temperature, and analyzed by SDS-PAGE and Western blotting. Mice were immunized intranasally with rSjFer, using chitosan as adjuvant. Two weeks after the third vaccination, challenge infection with S. japonicum cercariae was carried out. Worms and eggs collected from the livers of mice were counted at 42 days after the challenge. Levels of specific antibodies were detected by ELISA before infection., Results: SjFer was successfully subcloned into pTWIN1 vector and expressed in E. coli. In mice vaccinated intranasally with rSjFer and adjuvant chitosan, the worm reduction rate was 35.51% and the reduction rate of eggs per gram liver tissue (LEPG) was 52.17%. As compared with the control groups, levels of IgG, IgA in sera and SIgA in saliva increased significantly., Conclusion: The expressed rSjFer can induce partial protective immunity against S. japonicum infection in mice when they were vaccinated intranasally, with chitosan as adjuvant.

Objective: To determine the sensitivity of mouse to human cytomegalovirus AD169 strain (HCMV AD169) infection and to establish a stable animal model for developmental disorders., Methods: Forty kunmin mice were divided into two groups randomly: the virus inoculated group and the control group. After 7, 15, 30, and 60 days, the mice's brain development was observed by pathological techniques. HCMV antigen was detected by immunohistochemical method and HCMV DNA was analyzed by polymerase chain reaction (PCR)., Results: The extensive pathological damages and developmental disorders in the brain tissues of infected mice were observed. Meanwhile, the viral antigen and viral DNA were also demonstrated in the brain tissues of HCMV infected mice., Conclusion: HCMV can cause brain developmental disorders in mice, which provides a rationale for understanding the mechanism of congenital abnormality by HCMV infection.