Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay.

AIMS: Development of a multiplex TaqMan RT-qPCR assay to simultaneously detect Narcissus yellow stripe virus (NYSV) and Narcissus mosaic virus (NMV), frequently causing mixed narcissus infection. Feasibility verification was confirmed in natural samples. METHODS AND RESULTS: Primers and probes were designed based on the conserved CP gene regions of NYSV or NMV and their suitability for singleplex and multiplex TaqMan RT-qPCR assays as well as for conventional RT-PCR. Conventional RT-PCR, singleplex and multiplex TaqMan RT-qPCR assays proved to be NYSV and NMV specific. P-values and coefficients of variation of TaqMan RT-qPCR assays indicated high reproducibility. Significantly increased sensitivity was achieved compared to conventional RT-PCR. The detection limit of both viruses was 103 copies with superior correlation coefficients and linear standard curve responses between plasmid concentrations and Ct values. NYSV and NMV infection of narcissus leaves, petals and bulbs could successfully be detected via our multiplex RT-qPCR method at 1·25 mg. CONCLUSION: Our multiplex TaqMan RT-qPCR assay provides rapid, specific, sensitive and reliable testing to simultaneously detect NYSV and NMV, supplying useful routine monitoring for different narcissus samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient identification and discrimination of the narcissus viruses provides reliable information for scientists and conventional growers. Furthermore, it enriches the information of NYSV, NMV and other narcissus viruses.

HER 2/neu protein expression in gastric cancer is associated with poor survival.

Mol Med Rep 2: [Related article:] 943­946, 2009; DOI: 10.3892/mmr_00000196 After the publication of the article, the authors noted that there was an error regarding the author list on the Pubmed website. J.Y. Shen was erroneously omitted from the data submitted for publication on the Pubmed website. We apologize for the oversight and possible misunderstanding. The correct author list is the list that was published in PDF format on the Spandidos Publications website and in print, which is as follows: S.D. Xie1*, C.Y. Xu1,3*, J.G. Shen1, Z.N. Jiang2,3, J.Y. Shen1 and L.B. Wang1.

Self-priming on the plant viral RNAs during reverse transcription-PCR.

The occurrence of the primer-independent cDNA synthesis during RT-PCR analysis of human and animal RNA viruses has been well documented. Conversely, there is scant knowledge about this event in plant RNA viruses. Here we show that the primer-independent cDNA synthesis occurs in all eight different plant RNA viruses tested in this study, suggesting a common phenomenon for RT-PCR analysis of plant RNA viruses. Additional experiments indicate that the event is likely contributed to by RNA self-priming, and can be effectively reduced or eliminated through increasing temperature of the RT reaction.

[Analysis of tissue-specific differentially methylated genes with differential gene expression in non-small cell lung cancer].

Adenocarcinoma (ADC) and squamous cell carcinomas (SCC) are two subtypes of non-small cell lung carcinomas which are regarded as the leading cause of cancer-related malignancy worldwide. The aim of this study is to detect the differentially methylated loci (DMLs) and differentially methylated genes (DMGs) of these two tumor sets, and then to illustrate the different expression level of specific methylated genes. Using TCGA database and Illumina HumanMethylation 27 arrays, we first screened the DMGs and DMLs in tumor samples. Then, we explored the BiologicalProcess terms of hypermethylated and hypomethylated genes using Functional Gene Ontology (GO) catalogues. Hypermethylation intensively occurred in CpG-island, whereas hypomethylation was located in non-CpG-island. Most SCC and ADC hypermethylated genes involved GO function of DNA dependenit regulation of transcription, and hypomethylated genes mainly 'enriched in the term of immune responses. Additionally, the expression level of specific differentially methylated genesis distinctbetween ADC and SCC. It is concluded that ADC and SCC have different methylated status that might play an important role in carcinogenesis.

Prognostic improvement of reexcision for positive resection margins in patients with advanced gastric cancer.

AIM: The aim of this study was to investigate the effect of reexcision for advanced gastric cancer (GC) with positive resection margins on prognosis and to identify the selection criteria for the reexcision of patients with positive margins. PATIENTS AND METHODS: This was a retrospective study of 122 patients with positive margins who underwent potentially curative resection for locally advanced GC. The clinicopathological factors and survival among 50 patients who were reexcised to a negative resection margin (NR group) and 72 patients who were left with a positive resection margin (PR group) were compared using univariate and multivariate analyses. RESULTS: Median survival in the PR group was 18 months compared with 23 months in the NR group (p = 0.019). In the ≤ pN2-category subset, the PR group had a significantly worse prognosis compared with the NR group (median survival of 25 months vs. 44 months; p = 0.021). This difference was not observed in the pN3-category subset. In the univariate analysis, variables including pTNM stage, pN-category, and positive resection margin had adverse effects on OS among the entire population of 122 patients. A positive margin was confirmed as an independent prognostic factor for OS in the multivariate analysis. CONCLUSIONS: The reexcision of a positive margin improves the prognosis of patients with advanced GC, especially in those paitents with ≤ pN2-category disease and in patients undergoing D2 lymphadenectomy. Obtaining routine frozen sections of samples from the resection margin should be mandatory in the treatment of all GC patients undergoing potentially curative surgery.

Reduced lymph node harvest after neoadjuvant chemotherapy in gastric cancer.

This retrospective study investigated the impact of neoadjuvant chemotherapy on the number of lymph nodes harvested in patients with T(3)/T(4) gastric cancer. Lymph node counts in 58 patients who received preoperative neoadjuvant chemotherapy were compared with those in 168 patients who received surgery alone. Significantly more patients (n = 14, 24.1%) treated with neoadjuvant chemotherapy had < 15 lymph nodes harvested compared with patients (n = 13, 7.7%) treated with surgery alone. A significant correlation between the total number of harvested lymph nodes and the number of metastatic lymph nodes (mLNs) existed in both groups. Neoadjuvant chemotherapy was the only factor associated with the retrieval of < 15 lymph nodes. The number of mLNs was an independent predictive factor for overall survival. Although neoadjuvant chemotherapy decreased the number of lymph nodes harvested, the number of mLNs may still be an acceptable prognostic factor in patients with gastric cancer, following neoadjuvant chemotherapy.

Prognostic role of estrogen receptor alpha and estrogen receptor beta in gastric cancer.

BACKGROUND: The presence of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) have been reported in cell and tissue level in gastric cancer, but its impact on patients' survival remains unclear. This study was designed to investigate the expression level of ERalpha and ERbeta and to assess clinical significance of ERalpha and ERbeta expression in gastric cancer. METHODS: The expression level of ERalpha and ERbeta were assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) in 35 surgically resected gastric cancer and corresponding normal tissues and by immunohistochemical staining in 211 surgically resected gastric cancer and match normal tissues. RESULTS: The expression level between ERalpha mRNA expression in gastric cancer tissues and match normal tissues had no statistically significant difference. The ERbeta mRNA level in normal tissues was significantly higher than that observed in gastric cancer tissues (P = 0.001). Neither ERalpha nor ERbeta mRNA expression levels had significant correlation with clinicopathologic parameters. Forty-eight of 211 (22.7%) gastric cancer tissues showed positive expression of ERalpha and ERbeta detected in gastric cancer. ERalpha-positive expression correlated with poorer overall survival (P = 0.014), as did the absence of ERbeta expression in patients with gastric cancer (P = 0.001). In multivariate analysis, the positive expression of ERalpha and the absence of ERbeta were significant independent prognostic factors for overall survival (hazard ratio 2.159, P = 0.013, and hazard ratio 2.016, P = 0.025 respectively). CONCLUSIONS: Our results indicated that ERalpha and ERbeta were expressed in both gastric cancer and corresponding normal tissues. ERalpha expression and the absence of ERbeta expression are associated with poor survival.

Estrogen receptor beta variants mRNA expressions in gastric cancer tissues and association with clinicopathologic parameters.

BACKGROUNDS/AIMS: The aim of this study is to examine the expression of estrogen receptor beta-1, 2 ,5 in gastric cancer tissues and evaluate their correlation with clinicopathological features. METHODOLOGY: Real-time quantitative PCR assay was applied to detect the expression of estrogen receptor beta-1, 2, 5 mRNAs in 44 gastric cancer tissues and their paired normal tissues and correlate their mRNA levels with the clinicopathological properties of the tumors. 2(deltaCT) method was used to obtain the relative quantity of target mRNA expression. RESULTS: In almost all patients, estrogen receptor beta-1, 2, 5 mRNAs were expressed in gastric cancers and their matched normal tissues; however estrogen receptor beta-5 mRNA was not found in 8 normal gastric tissues. Estrogen receptor beta-5 had a much higher expression than estrogen receptor beta-1, 2 in gastric cancer tissues. Higher estrogen receptor beta-5 mRNAlevel was observed in gastric cancers than matched normal tissues (p = 0.001) and its increased expression was correlated with pTNM stage of the tumor (p = 0.032) and the lymph node metastasis (p = 0.026). Decreased mRNA level of estrogen receptor beta-1 was observed in gastric cancers compared to their matched normal tissues (p = 0.008). Estrogen receptor beta-1, 2 were not correlated with lymph node metastasis, gender, age, tumor size, tumor grade and pTNM stage (p > 0.05). CONCLUSIONS: This is the first study investigating the clinicopathologic role of estrogen receptor beta variants in gastric cancer. Our study shows that estrogen receptor beta-5 is the most important factor for gastric cancer development and progression among the three estrogen receptor beta variants.

Ulcer size as a novel indicator marker is correlated with prognosis of ulcerative gastric cancer.

AIMS: The objective of this study was to assess the significance of ulcer size for the survival of gastric cancer patients. METHODS: A total of 260 patients with ulcerative gastric cancer who had undergone curative resection were reviewed. The diameter of the malignant ulcer was measured. Patients were divided into group U1 (< or =3 cm) and group U2 (>3 cm) according to the diameter of the ulcer. The prognostic significance of ulcer size was assessed by uni- and multivariate analyses. RESULTS: Patient survival was correlated with age, gender, tumor location, tumor size, ulcer size, serosal invasion, node involvement and synchronism distant metastasis. The 5-year overall survival rate in U1 patients was 84.3% as compared with 67.5% in U2 patients (p < 0.001), and the 5-year recurrence-free survival rates were 82.9% for group U1 and 62.5% for group U2 (p = 0.001). Multivariate analysis revealed that ulcer size is an independently significant predictive factor for survival rates (overall: hazard ratio 1.222, p = 0.003; recurrence-free: hazard ratio 1.205, p = 0.006). CONCLUSION: Ulcer size might be a potential indicator for advanced disease and the use of minimal local treatments must be considered carefully in larger ulcer size patients.

HER 2/neu protein expression in gastric cancer is associated with poor survival.

The presence of HER 2/neu has been reported in gastric cancer, but its impact on patient survival remains unclear. The purpose of this study was to investigate the expression of HER 2/neu in gastric cancer. A total of 218 paired resected gastric cancer and corresponding normal specimens were collected. HER 2/neu protein expression was assessed by immunohistochemical staining. The correlation between HER 2/neu expression and patient clinicopathological parameters was evaluated and the prognostic significance of HER 2/neu expression was assessed by univariate and multivariate analyses. Forty-one out of 218 (18.8%) gastric cancer specimens showed HER 2/neu-positive expression. No relationship was found between membranous HER 2/neu expression and clinicopathological parameters. However, HER 2/neu expression was correlated with poorer overall survival (p<0.001). In multivariate analysis, HER 2/neu expression was a significant independent prognostic predictor of gastric cancer (p<0.001), and was associated with poor survival in gastric cancer patients. These data indicate that HER 2/neu may play a major role in the therapeutic management of gastric cancer.

Vaginal paraganglioma presenting as a gynecologic mass: case report.

Paragangliomas in the vagina are extremely rare. Unwitting surgical excision of a functional paraganglioma may precipitate life-threatening complications. We present a case of a 38-year-old woman with a vaginal mass 3.0 cm in diameter who experienced a hypertensive crisis during an unwitting attempted surgical excision of the vaginal mass. The diagnosis of a vaginal functional paraganglioma was then made based on to her 16-year history of paroxysmal headaches, chest distress, palpitation and elevated levels of urinary vannillylmandelic acid (VMA). Consequently, after thorough presurgical preparation, the patient again underwent excision of the vaginal mass uneventfully. She has been followed-up for three years since surgery without any evidence of recurrence. The clinical features and perioperative management of functional vaginal paraganglioma are described.

A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe array.

AIMS: To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. METHODS AND RESULTS: A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC-6 and -9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88.5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR-ahpC intergenic region (86.5%), whereas all 35 susceptible isolates identified showed a wild-type hybridization pattern. The sensitivity and specificity were 88.5% and 100% for rifampicin resistance, and 86.5% and 100% for isoniazid resistance, respectively. CONCLUSION: A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. SIGNIFICANCE AND IMPACT OF THE STUDY: This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated.

Novel sparse component analysis approach to free radical EPR spectra decomposition.

Free radicals play important roles in many physiological and pathological pathways in biological systems. These free radicals can be detected and quantified by their EPR spectra. The measured EPR spectra are often mixtures of pure spectra of several different free radicals and other chemicals. Blind source separation can be applied to estimate the pure spectra of interested free radicals. However, since the pure EPR spectra are often not independent of each other, the approach based on independent component analysis (ICA) cannot accurately extract the required spectra. In this paper, a novel sparse component analysis method for blind source separation, which exploits the sparsity of the EPR spectra, is presented to reliably extract the pure source spectra from their mixtures with high accuracy. This method has been applied to the analysis of EPR spectra of superoxide, hydroxyl, and nitric oxide free radicals, for both simulated data and real world ex vivo experiment. Compared to the traditional self-modeling method and our previous ICA-based blind source separation method, the proposed sparse component analysis approach gives much better results and can give perfect separation for mixtures of superoxide spectrum and hydroxyl spectrum in the ideal noise-free case. This method can also be used in other similar applications of quantitative spectroscopy analysis.

Free radical EPR spectroscopy analysis using blind source separation.

In this paper, we propose a novel approach for electron paramagnetic resonance (EPR) mixture spectra analysis based on blind source separation (BSS) technique. EPR spectrum of a free radical is often superimposed by overlapping spectra of other species. It is important and challenging to accurately identify and quantify the 'pure' spectra from such mixtures. In this study, an automated BSS method implementing independent component analysis is used to extract the components from mixed EPR spectra that contain overlapping components of different paramagnetic centers. To apply this method, there is no requirement to know the component spectra or the number of components in advance. The method is applied to analyze free radical EPR spectra which are collected from standard chemical system, cultured cell suspense, and ex vivo rat kidneys by spin trapping EPR technique. Results show that the BSS method proposed here is capable of identifying the component EPR spectra from mixtures with unknown compositions. The BSS technique can offer powerful aids in resolving spectral overlapping problems in general EPR spectroscopy analysis.

Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation.

The inhibitory effects of Chinonin, a natural antioxidant extracted from a Chinese medicine, on apoptotic and necrotic cell death of cardiomyocytes in hypoxia-reoxygenation process were observed in this study. The possible mechanisms of Chinonin on scavenging reactive oxygen species and regulating apoptotic related genes bcl-2 and p53 were also investigated. Neonatal rat cardiomyocytes were subjected to 24-h hypoxia and 4-h reoxygenation. Cell death was evaluated by DNA electrophoresis on agarose gel, cell death ELISA and annexin-V-FLUOS/propidium iodide (PI) double staining cytometry. Hypoxia caused the increase of apoptotic rates and the release of lactate dehydrogenase (LDH), while reoxygenation not only further increased the apoptotic rates and leakage of LDH, but also induced necrosis of cardiomyocytes. In addition, hypoxia increased the levels of NO(2)(-)/NO(3)(-) and thiobarbituric acid reacted substances (TBARS), while reoxygenation decreased NO(2)(-)/NO(3)(-), but further increased TBARS in the cultured media. Moreover, hypoxia up-regulated the expression levels of bcl-2 and p53 proteins, while reoxygenation down-regulated bcl-2 and further up-regulated p53. Chinonin significantly decreased the rates of apoptotic and necrotic cardiomyocytes, and inhibited the leakage of LDH. It also diminished NO(2)(-)/NO(3)(-) and TBARS, down-regulated the expression level of p53 protein, and up-regulated bcl-2 protein, respectively. The results suggest that Chinonin has preventive effects against apoptotic and necrotic cell death and its protective mechanisms are related to the antioxidant properties of scavenging nitric oxide and oxygen free radicals, and the modulating effects on the expression levels of bcl-2 and p53 proteins.

[Mechanisms of yiqi tongluo pill on oxygen free radicals, nitric oxide and fibrinolysis in coronary heart diseases and hypertension patients].

OBJECTIVE: To clarify the mechanism of Yiqi Tongluo Pill (YQTLP) in treating coronary heart disease and hypertension. METHODS: The clinical effects of YQTLP on 97 coronary heart diseases (CHD) and hypertension patients, in comparing to the 92 patients treated with nitroglycerin was investigated. The changes of plasma lipid peroxide (LPO), superoxide dismutase (SOD), tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) and nitric oxide (NO) were observed in the patients treated with YQTLP and nitroglycerin, which were compared to 30 healthy subjects. RESULTS: The levels of LPO and PAI increased and the levels of SOD, t-PA and NO decreased significantly in the patients. The clinical effects of YQTLP were better than that of nitroglycerin in the patients with unstable angina, while the effects of YQTLP were worse than that of nitroglycerin in the patients of hypertension and hypertension with CHD. YQTLP could decrease the levels of LPO and PAI and increase the levels of SOD, t-PA and NO significantly. The effects of YQTLP on t-PA and SOD were better than that of nitroglycerin. CONCLUSION: The protective mechanisms of YQTLP were related to inhibit lipid peroxidation, protect endothelium-derived relaxing factor and adjust the fibrinolytic activities.

Efficiency of Ginkgo biloba extract (EGb 761) in antioxidant protection against myocardial ischemia and reperfusion injury.

The cardio-protective mechanisms of EGb 761, an extract of Ginkgo biloba leaves, on myocardial ischemia-reperfusion injury were investigated using rabbits subjected to 30 minutes of regional cardiac ischemia and 120 min of reperfusion under anesthesia. Compared to the saline perfused group, EGb 761 treatment (10 mg/kg, injected into the coronary artery) significantly inhibited the increase in lipid peroxidation and maintained total and CuZn-SOD levels in both plasma and tissue during and at the end of reperfusion. Both the decrease in tissue type plasminogen activator (t-PA) and the increase in plasminogen activator inhibitor-1 (PAI-1) caused by ischemia-reperfusion were also significantly suppressed by EGb 761 treatment. Furthermore, the ultrastructure of the myocytes of the EGb 761 treated heart was slightly damaged after ischemia-reperfusion, while the control ischemic-reperfused hearts demonstrated severe histological damages such as swelling and vacuolization of the mitochondria. These results suggest that EGb 761 protects hearts by its antioxidant properties and by its ability to adjust fibrinolytic activity.