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A practical strategy for the synthesis of spiro[5.5]trienones-fused selenocyanates and spiro[4.5]trienones-fused selenocyanates through electrophilic selenocyanogen cyclization and dearomative spirocyclization is reported. This approach was conducted under mild conditions with broad substrate scope and good functional group tolerance. The utility of this procedure is exhibited in the late-stage functionalization of nature product and drug molecules.

Background:As one of the main blinding ocular diseases, corneal blindness resulted from neovascularization that disrupts the angiogenic privilege of corneal avascularity. Following neovascularization, inflammatory cells are infiltrating into cornea to strengthen corneal injury. How to maintain corneal angiogenic privilege to treat corneal disease has been investigated for decades.Methodology:Local administration of viral and non-viral-mediated anti-angiogenic factors reduces angiogenic protein expression in situ with limited or free of off-target effects upon gene delivery. Recently, Mesenchymal Stem Cells (MSCs) have been studied to treat corneal diseases. Once MSCs are manipulated to express certain genes of interest, they could achieve superior therapeutic efficacy after transplantation.Discussion:In the text, we first introduce the pathological development of corneal disease in the aspects of neovascularization and inflammation. We summarize how MSCs become an ideal candidate in cell therapy for treating injured cornea, focusing on cell biology, property and features. We provide an updated review of gene-based therapies in animals and preclinical studies in the aspects of controlling target gene expression, safety and efficacy. Gene transfer vectors are potent to induce candidate protein expression. Delivered by vectors, MSCs are equipped with certain characters by expressing a protein of interest, which facilitates better for MSC-mediated therapeutic intervention for the treatment of corneal disease.Conclusion:As the core of this review, we discuss how MSCs could be engineered to be vector system to achieve enhanced therapeutic efficiency after injection.

Houttuynia cordata polysaccharide (HCP) is extracted from Houttuynia cordata, a key traditional Chinese medicine. The study was to investigate the effects of HCP on intestinal barrier and microbiota in H1N1 virus infected mice. Mice were infected with H1N1 virus and orally administrated HCP at a dosage of 40 mg(kg−1(d−1. H1N1 infection caused pulmonary and intestinal injury and gut microbiota imbalance. HCP significantly suppressed the expression of hypoxia inducible factor-1α and decreased mucosubstances in goblet cells, but restored the level of zonula occludens-1 in intestine. HCP also reversed the composition change of intestinal microbiota caused by H1N1 infection, with significantly reduced relative abundances of Vibrio and Bacillus, the pathogenic bacterial genera. Furthermore, HCP rebalanced the gut microbiota and restored the intestinal homeostasis to some degree. The inhibition of inflammation was associated with the reduced level of Toll-like receptors and interleukin-1β in intestine, as well as the increased production of interleukin-10. Oral administration of HCP alleviated lung injury and intestinal dysfunction caused by H1N1 infection. HCP may gain systemic treatment by local acting on intestine and microbiota. This study proved the high-value application of HCP.

How cells drive the phospholipid signal response to heat stress (HS) to maintain cellular homeostasis is a fundamental issue in biology, but the regulatory mechanism of this fundamental process is unclear. Previous quantitative analyses of lipids showed that phosphatidylinositol (PI) accumulates after HS in Ganoderma lucidum, implying the inositol phospholipid signal may be associated with HS signal transduction. Here, we found that the PI-4-kinase and PI-4-phosphate-5-kinase activities are activated and that their lipid products PI-4-phosphate and PI-4,5-bisphosphate are increased under HS. Further experimental results showed that the cytosolic Ca2+ ([Ca2+ ]c ) and ganoderic acid (GA) contents induced by HS were decreased when cells were pretreated with Li+ , an inhibitor of inositol monophosphatase, and this decrease could be rescued by PI and PI-4-phosphate. Furthermore, inhibition of PI-4-kinases resulted in a decrease in the Ca2+ and GA contents under HS that could be rescued by PI-4-phosphate but not PI. However, the decrease in the Ca2+ and GA contents by silencing of PI-4-phosphate-5-kinase could not be rescued by PI-4-phosphate. Taken together, our study reveals the essential role of the step converting PI to PI-4-phosphate and then to PI-4,5-bisphosphate in [Ca2+ ]c signalling and GA biosynthesis under HS.

Ethnopharmacological relevance Hottuynia cordata is an important traditional Chinese medicine for the treatment of respiratory diseases including bacterial and viral infections. Polysaccharides isolated from Houttuynia cordata (HCP), as its main ingredients, have been demonstrated to ameliorate the LPS-induced acute lung injury in mice. The study aimed to determine the protective effects of HCP on multiple organ injury in influenza A virus (IAV) H1N1 infected mice and its primary mechanisms in anti-inflammation and immune regulation. Materials and methods Mice were inoculated with IAV H1N1 and then treated with 20 or 40 mg/kg/d of HCP for survival test and acute lung-gut injury test. Results The treatment with HCP resulted in an increase in the survival rate of H1N1 infected mice and the protection from lung and intestine injury, accompanied with the reduced virus replication. HCP markedly decreased the concentration of pulmonary proinflammatory cytokines/chemokines and the number of intestinal goblet cells, and strengthened the intestinal physical and immune barrier, according to the increase of sIgA and tight junction protein (ZO-1) in intestine. At the same time, the inhibition of inflammation in lung and gut was related to the suppressing of the expression of TLR4 and p-NFκB p65 in lung. Conclusions These results indicated that HCP ameliorated lung and intestine injury induced by IAV attack. The mechanisms were associated with inhibition of inflammation, protection of intestinal barrier and regulation of mucosal immunity, which may be related to the regulation of gut-lung axis. As an alternative medicine, HCP may have clinical potential to treat IAV infection in human beings.

Bacterial lipopolysaccharide (LPS) contributes to airway inflammation and mucus hypersecretion in chronic airway inflammatory diseases, such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Neutrophil extracellular traps (NETs) are extracellular meshworks composed of DNA fibers and antimicrobial proteins. Although NET formation has been detected in COPD and CF patients, how NETs contribute to these diseases is poorly understood. This study was performed to clarify the effects and mechanisms of action of NETs in airway inflammation and mucus hypersecretion. We created a murine model of LPS-induced airway inflammation and mucus hypersecretion, and found that LPS-induced NET formation was degraded by aerosolized DNase I treatment in mice. Degradation of NETs by aerosolized DNase I reduced LPS-induced airway inflammation and mucus hypersecretion in mice, this reduction correlated with suppression of TLR4/NF-κB signaling pathway. More importantly, NETs promoted LPS-induced production of IL-1β, IL-6 and TNF-α in macrophages. These results suggest NET degradation using aerosolized DNase I is a potential new therapeutic strategy for treating COPD and CF.

Phospholipid-mediated signal transduction plays a key role in responses to environmental changes, but little is known about the role of phospholipid signalling in microorganisms. Heat stress (HS) is one of the most important environmental factors. Our previous study found that HS could induce the biosynthesis of the secondary metabolites, ganoderic acids (GA). Here, we performed a comprehensive mass spectrometry-based analysis to investigate HS-induced lipid remodelling in Ganoderma lucidum. In particular, we observed a significant accumulation of phosphatidic acid (PA) on HS. Further genetic tests in which pld-silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS-activated phospholipase D (PLD) hydrolysing phosphatidylethanolamine. Furthermore, we determined the role of PLD and PA in HS-induced secondary metabolism in G. lucidum. Exogenous 1-butanol, which decreased PLD-mediated formation of PA, reverses the increased GA biosynthesis that was elicited by HS. The pld-silenced strains partly blocked HS-induced GA biosynthesis, and this block can be reversed by adding PA. Taken together, our results suggest that PLD and PA are involved in the regulation of HS-induced secondary metabolism in G. lucidum. Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

Ceramide synthases (CERSs) catalyse an N-acyltransferase reaction using long-chain base (LCB) and fatty acyl-coenzyme A (CoA) as substrates to synthesize ceramide (Cer), which is the backbone of all complex sphingolipids. In the present study, three CERSs (LAG1, LAG2 and LAG3) form Ganoderma lucidum were analysed. The silencing of lag1 by RNA interference reduced ganoderic acid biosynthesis and Cer and complex sphingolipids contents, which contain long-chain-fatty-acids (LCFAs, including C16 and C18). In contrast, the silencing of lag2 or lag3 did not result in obvious phenotypic and sphingolipid homeostasis changes, although the lag2/lag3 double-silenced mutants exhibited increased ganoderic acid biosynthesis as well as reduced growth, reduced Cer and complex sphingolipids contents, which contain very-long-chain fatty acids (VLCFAs, including C22, C24 and C26). The results of the present study indicate that the three assayed CERSs have distinct physiological functions and substrate specificities in G. lucidum.

The three-dimensional solid model of the chassis which is the support structure for the wheeled robot's load and power parts is established. Based on the theory of elasticity, the relation equations of the three direction stress and strain of the chassis structure are given. The calculation analysis and simulation are completed by the finite element method, and the structural stress and strain of the chassis under loads and driving forces are obtained. Through simulating and analyzing the stress, strain and deformation values and distribution on the chassis structure, it makes a basis for the design and research of the chassis with light weighted and appropriate stress and deformation.

This study aimed to identify suitable reference genes under three chemical inducers, methyl jasmonate (MeJA), salicylic acid (SA) and hydrogen peroxide (H2O2) in Ganoderma lucidum. In this study, expression stabilities of 14 candidate reference genes had been validated. Four algorithms were used: geNorm, NormFinder, BestKeeper, and RefFinder. Our results showed that, in short time, UCE2 (ubiquitin conjugating enzyme) was the most stable gene both in MeJA and H2O2 treatments, ACTIN (beta-actin) was the most suitable reference gene for SA treatment. ACTIN/UCE2 were considered the most suitable genes to normalize in MeJA, SA and H2O2 conditions. In long time, PP2A (protein phosphatase 2A regulatory subunit) was the most stable gene in MeJA and SA treatments, UCE2 was the most suitable reference gene for H2O2 treatment. PP2A/UBQ1 (polyubiquitin 1) were considered the most suitable genes to normalize in MeJA, SA and H2O2 conditions. Furthermore, target gene, oxidosqualene cyclase (osc), was selected to validate the most and least stable reference genes under different treatments. Our work provided a better support to study the regulatory mechanism of MeJA, SA and H2O2 on biological functions.

A device was designed to form the shaped charge jets without slug based on the structure of 56mm shaped charge, and the movement of slug was under controlled through the use of this device. A numerical simulation of the shaping of jets under the structure of shaped charge was carried out by LS-DYNA. On the premise of the device wall thickness 1mm, the angle of middle part and the height of the lower part, here the 2 structural parameters of the blocking slug device are considered as the factors affecting the shaping of the jets without slug. Through simulation, the best structural parameters of the device are as follows: 45 degree of the inclination of the middle part and 0mm of lower part. Under this structural parameter, the device can make shaped charge fully molded without slug.

How cells drive the phospholipid signal response to heat stress (HS) to maintain cellular homeostasis is a fundamental issue in biology, but the regulatory mechanism of this fundamental process is unclear. Previous quantitative analyses of lipids showed that phosphatidylinositol (PI) accumulates after HS in Ganoderma lucidum, implying the inositol phospholipid signal may be associated with HS signal transduction. Here, we found that the PI-4-kinase and PI-4-phosphate-5-kinase activities are activated and that their lipid products PI-4-phosphate and PI-4,5-bisphosphate are increased under HS. Further experimental results showed that the cytosolic Ca

Ganoderma lucidum has been considered an emerging model species for studying how environmental factors regulate the growth, development, and secondary metabolism of Basidiomycetes. Heat stress, which is one of the most important environmental abiotic stresses, seriously affects the growth, development, and yield of microorganisms. Understanding the response to heat stress has gradually become a hotspot in microorganism research. But suitable reference genes for expression analysis under heat stress have not been reported in G. lucidum. In this study, we systematically identified 11 candidate reference genes that were measured using reverse transcriptase quantitative polymerase chain reaction, and the gene expression stability was analyzed under heat stress conditions using geNorm and NormFinder. The results show that 5 reference genes-CYP and TIF, followed by UCE2, ACTIN, and UBQ1-are the most stable genes under our experimental conditions. Moreover, the relative expression levels of 3 heat stress response genes (hsp17.4, hsp70, and hsp90) were analyzed under heat stress conditions with different normalization strategies. The results show that use of a gene with unstable expression (SAND) as the reference gene leads to biased data and misinterpretations of the target gene expression level under heat stress.

Ethnopharmacological relevance Houttuynia cordata (HC) has been used as a folk therapy to treat pulmonary infections. This study aimed to determine the role and mechanism of action of polysaccharides isolated from HC (HCP) in lipopolysaccharide (LPS)-induced ALI in the mice. Materials and methods LPS was delivered by the intratracheal route to Balb/c mice 2 h before HCP (40, 80 and 160 mg/kg) administration. Results The number of total cells, protein and tumor necrosis factor-α (TNF-α) concentrations in bronchoalveolar lavage fluid, the wet/dry weight ratio (w/d) of lungs and pulmonary pathology of each mouse were analyzed, it was found that HCP significantly alleviated ALI induced by LPS. Moreover, in lungs of mice, it was found that the infiltration of inflammatory cells, the expression of Toll-like receptor 4 and complement deposition were significantly decreased by HCP treatment. In vitro assays showed that C5a, a complement activation product, induced significant macrophage migration and treatment with HCP prevented it. The in vitro results also proved that LPS increased nitric oxide and pro-inflammatory cytokines (TNF-α, interleukin-6, and interleukin-1β) production, and HCP antagonized these effects of LPS. It was also found that HCP alone augmented secretion of some pro-inflammatory cytokines. Conclusion These results indicate that HCP may alleviate LPS induced lung inflammatory injury, which may be associated with its inhibitory effect on the over activation of complement and macrophages. This suggests a potential role to treat ALI., Graphical abstract

// Zhenghu Li 1 , Zhi-Qiang Ling 2 , Weiwei Guo 1 , Xiao-Xiao Lu 2 , Yi Pan 1 , Zhenzhen Wang 1 , Yan Chen 1 1 Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China 2 Zhejiang Cancer Research Institute, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, China Correspondence to: Yan Chen, email: // Zhi-Qiang Ling, email: // Keywords : breast cancer, PAQR3, HER2, survival, cell proliferation Received : February 02, 2015 Accepted : February 28, 2015 Published : March 26, 2015 Abstract PAQR3 is a newly discovered tumor suppressor and its functional role in breast cancer has not been well characterized. We report here that PAQR3 is associated with the progression and survival of human patients with breast cancer, as well as cell proliferation and migration of human breast cancer cells. PAQR3 mRNA level was robustly downregulated in human breast cancer samples compared with their corresponding para-cancerous histological normal tissues ( n = 82, P < 0.0001). The mRNA level of PAQR3 was negatively correlated with HER2 expression ( P < 0.0001) and disease-free survival of the patients ( P < 0.0001). PAQR3 overexpression inhibited cell proliferation, colony formation and migration of breast cancer cells including MCF7, SKBR3, MDA-MD-231, MDA-MD-468 and MDA-MD-453 cells. Knockdown of PAQR3 in MDA-MD-231 cells elevated cell proliferation and migration. Inhibition of HER2 by trastuzumab increased PAQR3 expression in SKBR3 cells. In conclusion, PAQR3 expression is frequently downregulated in human breast cancers inversely correlated with HER2 expression. PAQR3 is able to modulate the proliferation and migration of breast cancer cells. Our data indicate that PAQR3 functions as a tumor suppressor in the development of human breast cancers.

Phospholipid-mediated signal transduction plays a key role in responses to environmental changes, but little is known about the role of phospholipid signalling in microorganisms. Heat stress (HS) is one of the most important environmental factors. Our previous study found that HS could induce the biosynthesis of the secondary metabolites, ganoderic acids (GA). Here, we performed a comprehensive mass spectrometry-based analysis to investigate HS-induced lipid remodelling in Ganoderma lucidum. In particular, we observed a significant accumulation of phosphatidic acid (PA) on HS. Further genetic tests in which pld-silencing strains were constructed demonstrated that the accumulation of PA is dependent on HS-activated phospholipase D (PLD) hydrolysing phosphatidylethanolamine. Furthermore, we determined the role of PLD and PA in HS-induced secondary metabolism in G. lucidum. Exogenous 1-butanol, which decreased PLD-mediated formation of PA, reverses the increased GA biosynthesis that was elicited by HS. The pld-silenced strains partly blocked HS-induced GA biosynthesis, and this block can be reversed by adding PA. Taken together, our results suggest that PLD and PA are involved in the regulation of HS-induced secondary metabolism in G. lucidum. Our findings provide key insights into how microorganisms respond to heat stress and then consequently accumulate secondary metabolites by phospholipid remodelling.

Background.Papillary thyroid carcinoma (PTC) is a form of thyroid cancer with high risk of cervical lymph node metastasis.Aim.The aim of this study was to investigate the incidence and the predictive factors for occult ipsilateral central lymph node (CLN) metastasis in the patients with papillary thyroid carcinoma.Methods.A total of 916 PTC patients (1017 lesions) undergoing central lymph node dissection in our hospital from 2005 to 2011 were enrolled. The relationship between CLN metastasis and clinical factors such as gender, age, tumor size, tumor number, capsule invasion, and tumor location was analyzed.Results.Occult CLN metastasis was observed in 52.41% (533/1017) of PTC lesions, respectively. Multivariate analysis showed that age ≤ 35 years, tumor size > 1.5 cm, present capsule invasion/extracapsular invasion, and tumor located in upper/middle pole/whole lobe were risk factors of CLN metastasis.Conclusions.Tumor located in upper/middle pole/whole lobe, less than 35 years old, tumor size > 1.5 cm, and present capsule invasion/extracapsular invasion were risk factors of CLN metastasis. We recommend performing ipsilateral prophylactic CLN dissection in cN0 PTC patients.

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

Lipopolysaccharides (LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclear cells. LPS was pretreated with polymyxin B sulfate (PB) at different concentrations for 1, 5 or 24 h, and then used to stimulate RAW264.7 and mouse peritoneal macrophages (MPMs). The nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in cell culture supernatant, as the indications of cell response, were assayed. Bupleurum chinensis polysaccharides (BCPs) with trace amount contamination of LPS was treated with PB. 30 μg·mL-1 of PB, treating LPS (10 and 1 000 ng·mL-1 in stimulating RAW264.7 and MPMs respectively) at 37 °C for 24 h, successfully abolished the stimulating effect of LPS on the cells. When the cells were stimulated with LPS, BCPs further promoted NO production. However, pretreated with PB, BCPs showed a suppression of NO production in MPMs and no change in RAW264.7. In the in vitro experiments, LPS contamination in polysaccharide might bring a great interference in assessing the activity of drug. Pretreatment with PB (30 μg·mL-1) at 37 °C for 24 h was sufficient to abolish the effects of LPS contamination (10 and 1 000 ng·mL-1).

The involvement of the receptor for advanced glycation end (RAGE) in different diseases has been reviewed in great detail, previously, but the effects of diabetic drugs on RAGE-induced skin lesion during long course diabetes remains poorly understood. In the present study, we have shown that RAGE was overexpressed in both diabetic rats and human keratinocytes (HaCaT cells). Cell cycle arrest and apoptosis as well as alternations of relative protein levels were also found in diabetic rats and HaCaT cells with overexpression of RAGE that were rectified by metformin (Met) treatment. Moreover, overexpression of RAGE was also found to induce secretions of TNF-α, IL-1β, IL-6, ICAM-1 and COX-2 in HaCaT cells, and Met treatment corrected these inflammatory factor secretions. In addition, treatment with Met markedly reduced RAGE overexpression-induced p38 and NF-κB activation. Taken together, the findings of the present study have demonstrated, for the first time that Met protects HaCaT cells against diabetes-induced injuries and inflammatory responses through inhibiting activated RAGE.

Diabetes mellitus is related to low-grade chronic inflammation and oxidative stress. Bupleurum Polysaccharides (BPs), isolated from Bupleurum smithii var. parvifolium has anti-inflammatory and anti-oxidative properties. However, little is known about its therapeutic effects on diabetes. In this experiment, the effects of BPs on alleviation of diabetes and the underlying mechanisms were investigated. Diabetic mice model was established via successive intraperitoneal injections of streptozotocin (100 mg/kg body weight) for two days. Mice with blood glucose levels higher than 16.8mmol/L were selected for experiments. The diabetic mice were orally administered with BPs (30 and 60 mg/kg) once a day for 35 days. BPs not only significantly decreased levels of blood glucose, but also increased those of serum insulin and liver glycogen in diabetic mice compared to model mice. Additionally, BPs adminstration improved the insulin expression and suppressed the apoptosis in pancreas of the diabetic mice. Histopathological observations further demonstrated that BPs protected the pancreas and liver from oxidative and inflammatory damages. These results suggest that BPs protect pancreatic β cells and liver hepatocytes and ameliorate diabetes, which is associated with its anti-oxidative and anti-inflammatory properties.

BACKGROUND H2AX, one of the variants of histone H2A with conserved sequence, plays a vital role in maintaining genomic stability. DNA double-strand break (DSB) induces H2AX phosphorylation (γH2AX). Growing evidence indicated γH2AX was linked to thyroid disease. However, the precise expression of γH2AX in papillary thyroid carcinoma (PTC) and nodular goiter was unknown. In this study, the yH2AX expression in PTC and nodular goiter and the significance of the aberrant expression were investigated. METHODS The γH2AX expression level was evaluated by immunohistochemistry staining in 140 specimens with thyroid diseases (30 cases of nodular goiter, 30 cases of PTC, and 80 cases of PTC coexisting with nodular goiter). The possible relationships between γH2AX expression and patients' clinicopathological characteristics were analyzed. RESULTS In the set of nodular goiter, 20.0% (6/30) of nodular goiter component and 10.0% (3/30) of normal adjacent tissues exhibited high expression of γH2AX (p > 0.05). In the set of PTC, 70.0% (21/30) of PTC component and 0.0% (0/30) of normal adjacent tissues showed high expression of γH2AX (p < 0.05). In the set of PTC coexisting with nodular goiter, 95.0% (76/80) of PTC component, 12.5% (10/80) of nodular goiter component, and 8.8% (7/80) of normal adjacent specimens showed high expression of γH2AX (p < 0.05). 85.5% (65/76) of PTC patients with lymph node metastasis were found to have a high expression level of γH2AX, and the proportion was lower than that in patients without lymph node metastasis (34/34, 100.0%) (p = 0.003). High γH2AX expression was also found in 76.2% (16/21) of stage IV patients and the proportion was lower than that in stage I - III patients (83/89, 93.3%) (p = 0.034). No statistically significant correlation was found between yH2AX expression level and other clinicopathological features, including age, gender, capsular invasion, tumor size, tumor focus number, distant metastasis, and T status. CONCLUSIONS γH2AX expression was increased in PTC patients, while nodular goiter and normal adjacent tissues exhibited no statistically significant difference in γH2AX expression. yH2AX expression had a negative correlation with TNM stage and lymph node metastasis. γH2AX seems to play a more critical role in early-stage PTC rather than late-stage PTC and might inhibit lymph node metastasis.

N-myc downstream regulated gene 2 (Ndrg2) is a candidate suppressor of cancer metastasis. We found that Ndrg2 promoter was frequently hypermethylated in gastric cancer cell lines and in 292 gastric tumor tissues. This resulted in down-regulation of Ndrg2 mRNA and protein. Ndrg2 promoter methylation was associated with H. pylori infection and worse prognosis of gastric cancer patients, which is an independent prognostic factor for the disease-free survival (DFS). We found that H. pylori silenced Ndrg2 by activating the NF-κB pathway and up-regulating DNMT3b, promoting gastric cancer progression. These findings uncover a previously unrecognized role for H. pylori infection in gastric cancer.

To investigate the aberrant promoter methylation of hMLH1 gene promoter and its clinical significance in papillary thyroid cancer (PTC).methylation of hMLH1 gene promoter in the cancer tissue and matched tumor-adjacent normal tissue of 152 PTC patients were detected by real-time methylation specific PCR (qMSP). The relationship between the methylation of hMLH1 gene promoter and clinicopathological features was analyzed.The methylation rate of hMLH1 gene promoter in cancer tissues was 37.5% (57/152), of which 33 cases were totally methylated and 24 cases were partially methylated. The methylation rate of adjacent normal tissues was 5.3% (8/152)(all were partially methylated). The methylation rate of PTC tissues was significantly higher than that in the tumor-adjacent normal tissue (P0.01). The promoter methylation of hMLH1 gene in PTC was significantly correlated with age, size and number of the primary lesion, local invasion, T stage and lymph node metastasis (P0.05) , but not correlated with gender and clinical stage (P0.05).Promoter methylation of hMLH1 gene is a common molecular event in PTC tissue, and it is significantly correlated with the progression of PTC.

RegIV, a member of the Regenerating (REG) gene family, may be a marker for the prediction of resistance to 5-fluorouracil (5-FU)-based chemotherapy. However, the relationship between the intrinsic drug resistance of gastric cancer (GC) cells to 5-FU used alone (single FU) or in multidrug therapeutic regimens (5-FU combinations) and RegIV expression has not been investigated. The patient cohort comprised 45 patients with primary GC. The chemoresistance of GC cells to therapeutic regimens consisting of single 5-FU or FU combinations was investigated using the ATP-tumor chemosensitivity assay. The level of RegIV mRNA transcripts was determined by real-time reverse transcriptase-PCR. RegIV expression was evaluated as a novel predictive biomarker for the intrinsic drug resistance of primary GC cells to single 5-FU or 5-FU combinations. Upregulation of RegIV mRNA transcripts was observed in 36 of the 45 tumor specimens and was positively correlated with the invasive depth of the tumor cells (p = 0.000), the clinical stages (p = 0.000) and the in vitro intrinsic drug resistance of primary GC cells to 5-FU (p = 0.000) or 5-FU combinations. RegIV mRNA transcript level was strongly associated with the intrinsic resistance of GC cells to single 5-FU or 5-FU combinations, suggesting that RegIV may play an important role in the intrinsic resistance of GC cells to 5-FU and that targeted therapy against the RegIV gene could be applied to overcome 5-FU resistance in the treatment of GC.

Current chemotherapy for esophageal cancer is conducted on the basis of empirical information from clinical trials, which fails to take into account the known heterogeneity of chemosensitivity between patients. This study was aimed to demonstrate the degree of heterogeneity of chemosensitivity in esophageal cancers.A total of 42 esophageal cancer specimens were collected. The heterogeneity of chemosensitivity in esophageal cancer specimens was examined using an ex vivo ATP-tumor chemosensitivity assay (ATP-TCA).Thirty eight specimens produced evaluable results (90.5%). The most active single agent tested was nedaplatin, to which 28.9% of samples were sensitive. Combinations of chemotherapy agents exhibited much higher sensitivity: cisplatin + paclitaxel was sensitive in 16 of 38 (42.1%) of samples, while nedaplatin+paclitaxel was more effective, which was sensitive in 20 of 38 cases (52.6%).There was a marked heterogeneity of chemosensitivity in esophageal cancer. Chemosensitivity testing may provide a practical method for testing new regimens before clinical trials in esophageal cancer patients.

BACKGROUND: Helicobacter pylori has been recognized as a definite carcinogen for gastric cancer (GC); however, the pathogenesis of H. pylori infection remains unclear. Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene whose deficiency is causally related to GC. However, in H. pylori infection-associated GC, the role of RUNX3 has not been studied. METHODS: The authors used real-time methylation-specific polymerase chain reaction analysis to determine methylation status of the RUNX3 promoter in a spectrum of gastric lesions, including 220 samples of chronic atrophic gastritis, 196 samples of intestinal metaplasia, 134 samples of gastric adenoma, 102 samples of dysplasia, and 202 samples of GC with paired noncancerous mucosa tissues and corresponding blood specimens. The association of abnormal methylation with precancerous gastric lesions was evaluated along with the association between RUNX3 methylation and H. pylori infection, and the concordance of methylation levels was investigated between serum and tissues. RESULTS: The results indicated that increasing RUNX3 promoter methylation was correlated with distinct stages of GC progression. GC tissues had the highest methylation proportion (75.2%) compared with precancerous gastric lesions, including chronic atrophic gastritis (15.9%), intestinal metaplasia (36.7%), gastric adenoma (41.8%), and dysplasia (54.9%). H. pylori infection, a major risk factor for GC, contributed to the inactivation of RUNX3 in gastric epithelial cells through promoter hypermethylation. The levels of RUNX3 methylation in serum were in significant concordance with the methylation levels observed in GC tissues (P = .887). CONCLUSIONS: The current findings supported RUNX3 methylation as a risk factors for the carcinogenesis of chronic atrophic gastritis with H. pylori infection and indicated that circulating RUNX3 methylation is a valuable biomarker for the detection of early GC. Cancer 2012. © 2012 American Cancer Society.

e22042 Background: N-myc downstream regulated gene 2 (Ndrg2) promoter methylation was recently shown to be frequent in human cancers and was suggested as a candidate suppressor of cancer metastasis...

Tooth aspiration is a critical and rare complication in trauma patients. It can remain undetected and misdiagnosed. Here we present three mechanically ventilated multiple trauma cases with delayed diagnosis of bronchial tooth aspiration. Case 1 A 23-year-old man was severely injured in a car accident and had multiple trauma, with an injury severity score (ISS) of 50 and a head abbreviated injury score (AIS) of 5. Three days later he was transferred to our hospital and chest CT showed a tooth-like foreign body in the right bronchial tree, which was confirmed to be a bicuspid ( according to the Palmer notation method) by flexible bronchoscopy. As the tooth was psilate and firmly embedded, many attempts with either flexible or rigid bronchoscopy could not extract it. The patient died of sepsis 70 days after the accident. Case 2 A 26-year-old man was presented to a local hospital with multiple trauma (ISS 54, head AIS 5) following a motorcycle accident. On the next day when he was transferred to our hospital, a chest film and fibreoptic bronchoscopy confirmed a lateral incisor () in the left lobe. We failed to extract the tooth, but fortunately it was expectorated via the tracheotomy tube following a vigorous cough on day 12. Case 3 A 25-year-old man was presented with multiple trauma (ISS 41, head AIS 4) after a motorcycle accident. He was transferred to our hospital 9 hours later. Chest CT and flexible bronchoscopy confirmed a central incisor () in the right bronchial tree. It was extracted by flexible bronchoscopy on day 8 (Figure ​(Figure11). Figure 1 CT images of lung and maxillofacial bones, inspired tooth in case 3. (a-d) CT scan on day 1 showed the tooth-like foreign body (white/black arrow) in the right bronchus. (e) Reconstructed CT images on day 2 showed fracture of mandible and left zygomatic ... Early diagnosis of tooth aspiration in coma patients is difficult because aspiration is rarely considered in the absence of an acute clinical presentation. Thus, an endobronchial tooth can remain undetected for a long time and result in serious complications [1]. In all three cases reported here, tooth aspiration was missed by the local hospitals. Doctors should suspect that any tooth that has been avulsed and not found as possibly aspirated in trauma patients, especially for those in a coma status. The medical history, clinical signs and radiological findings should be carefully checked. CT is more accurate than chest radiography in detecting endobronchial foreign bodies. When negative chest radiography or doubtful clinical findings are presented, bronchoscopy should be performed to confirm the diagnosis [2]. Both flexible and rigid bronchoscopy can be used for the diagnosis and removal of teeth [3]. Tracheotomy may facilitate the removal of an aspirated tooth.