FGFR2 amplification has prognostic significance in gastric cancer: results from a large international multicentre study.

BACKGROUND: In preclinical gastric cancer (GC) models, FGFR2 amplification was associated with increased tumour cell proliferation and survival, and drugs targeting this pathway are now in clinical trials. METHODS: FGFR2 FISH was performed on 961 GCs from the United Kingdom, China and Korea, and the relationship with clinicopathological data and overlap with HER2 amplification were analysed. RESULTS: The prevalence of FGFR2 amplification was similar between the three cohorts (UK 7.4%, China 4.6% and Korea 4.2%), and intratumoral heterogeneity was observed in 24% of FGFR2 amplified cases. FGFR2 amplification was associated with lymph node metastases (P<0.0001). FGFR2 amplification and polysomy were associated with poor overall survival (OS) in the Korean (OS: 1.83 vs 6.17 years, P=0.0073) and UK (OS: 0.45 vs 1.9 years, P<0.0001) cohorts, and FGFR2 amplification was an independent marker of poor survival in the UK cohort (P=0.0002). Co-amplification of FGFR2 and HER2 was rare, and when high-level amplifications did co-occur these were detected in distinct areas of the tumour. CONCLUSION: A similar incidence of FGFR2 amplification was found in Asian and UK GCs and was associated with lymphatic invasion and poor prognosis. This study also shows that HER2 and FGFR2 amplifications are mostly exclusive.

Genome-wide association analysis identifies quantitative trait loci for growth in a Landrace purebred population.

Growth-related traits are complex and economically important in the livestock industry. The aim of this study was to identify quantitative trait loci (QTL) and the associated positional candidate genes affecting growth in pigs. A genome-wide association study (GWAS) was performed using the porcine single-nucleotide polymorphism (SNP) 60K bead chip. A mixed-effects model and linear regression approach were used for the GWAS. The data used in the study included 490 purebred Landrace pigs. All experimental animals were genotyped with 39 438 SNPs located throughout the pig autosomes. We identified a strong association between a SNP marker on chromosome 16 and body weight at 71 days of age (ALGA0092396, P = 5.35 × 10(-9) , Bonferroni adjusted P < 0.05). The SNP marker was located near the genomic region containing IRX4, which encodes iroquois homeobox 4. This SNP marker could be useful in the selective breeding program after validating its effect on other populations.

Genome-wide association study identifies quantitative trait loci affecting hematological traits in an F2 intercross between Landrace and Korean native pigs.

Changes affecting the status of health and robustness can bring about physiological alterations including hematological parameters in swine. To identify quantitative trait loci (QTL) associated with eight hematological traits (one leukocyte trait, six erythrocyte traits and one platelet trait), we conducted a genome-wide association study using the PorcineSNP60K BeadChip in a resource population derived from an intercross between Landrace and Korean native pigs. A total of 36 740 SNPs from 816 F2 progeny were analyzed for each blood-related trait after filtering for quality control. Data were analyzed by the genome-wide rapid association using mixed model and regression (GRAMMAR) approach. A total of 257 significant SNPs (P < 1.36 × 10(-6) ) on SSC3, 6, 8, 13 and 17 were identified for blood-related traits in this study. Interestingly, the genomic region between 17.9 and 130 Mb on SSC8 was found to be significantly associated with red blood cell, mean corpuscular volume and mean corpuscular hemoglobin. Our results include the identification of five significant SNPs within five candidate genes (KIT, IL15, TXK, ARAP2 and ERG) for hematopoiesis. Further validation of these identified SNPs could give valuable information for understanding the variation of hematological traits in pigs.

QTL analysis of body weight and carcass body length traits in an F2 intercross between Landrace and Korean native pigs.

Growth traits, such as body weight and carcass body length, directly affect productivity and economic efficiency in the livestock industry. We performed a genome-wide linkage analysis to detect the quantitative trait loci (QTL) that affect body weight, growth curve parameters and carcass body length in an F2 intercross between Landrace and Korean native pigs. Eight phenotypes related to growth were measured in approximately 1000 F2 progeny. All experimental animals were subjected to genotypic analysis using 173 microsatellite markers located throughout the pig genome. The least squares regression approach was used to conduct the QTL analysis. For body weight traits, we mapped 16 genome-wide significant QTL on SSC1, 3, 5, 6, 8, 9 and 12 as well as 22 suggestive QTL on SSC2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16 and 17. On SSC12, we identified a major QTL affecting body weight at 140 days of age that accounted for 4.3% of the phenotypic variance, which was the highest test statistic (F-ratio = 45.6 under the additive model, nominal P = 2.4 × 10(-11) ) observed in this study. We also showed that there were significant QTL on SSC2, 5, 7, 8, 9 and 12 affecting carcass body length and growth curve parameters. Interestingly, the QTL on SSC2, 3, 5, 6, 8, 9, 10, 12 and 17 influencing the growth-related traits showed an obvious trend for co-localization. In conclusion, the identified QTL may play an important role in investigating the genetic structure underlying the phenotypic variation of growth in pigs.

MET in gastric carcinomas: comparison between protein expression and gene copy number and impact on clinical outcome.

BACKGROUND: The aim of this study was to compare gene copy number (GCN) and protein expression of MET and to evaluate their prognostic roles in gastric carcinomas. METHODS: MET protein expression and gene amplification (GA) status were determined by immunohistochemistry (IHC) and silver in-situ hybridisation (SISH), respectively, in a large series of gastric carcinoma. RESULTS: Protein overexpression was observed in 104 of 438 cases, with IHC 2+ in 94 and IHC 3+ in 10, and high polysomy of chromosome 7 and GA were found in 61 and 13 of 381, respectively. Direct comparison revealed a significant correlation between high level of protein expression and increased GCN. All cases with GA showed protein overexpression. Furthermore, all with IHC 3+ showed GA except 1, even which could be categorised as GA according to the ASCO/CAP guideline for human epidermal growth factor receptor 2 assessment. IHC 3+ and GA were significantly associated with poor prognosis. CONCLUSION: MET IHC reflects well on GA, and therefore, it could be a primary screening test for patient selection for anti-MET therapy if GA is a major determinant of drug responsiveness. Also, the prognostic role of MET indicates that anti-MET therapy is a very promising modality in adjuvant treatment for gastric cancer.

QTL analysis of white blood cell, platelet and red blood cell-related traits in an F2 intercross between Landrace and Korean native pigs.

Haematological traits play important roles in disease resistance and defence functions. The objective of this study was to locate quantitative trait loci (QTL) and the associated positional candidate genes influencing haematological traits in an F(2) intercross between Landrace and Korean native pigs. Eight blood-related traits (six erythrocyte traits, one leucocyte trait and one platelet trait) were measured in 816 F(2) progeny. All experimental animals were genotyped with 173 informative microsatellite markers located throughout the pig genome. We report that nine chromosomes harboured QTL for the baseline blood parameters: genomic regions on SSC 1, 4, 5, 6, 8, 9, 11, 13 and 17. Eight of twenty identified QTL reached genome-wide significance. In addition, we evaluated the KIT locus, an obvious candidate gene locus affecting variation in blood-related traits. Using dense single nucleotide polymorphism marker data on SSC 8 and the marker-assisted association test, the strong association of the KIT locus with blood phenotypes was confirmed. In conclusion, our study identified both previously reported and novel QTL affecting baseline haematological parameters in pigs. Additionally, the positional candidate genes identified here could play an important role in elucidating the genetic architecture of haematological phenotype variation in swine and in humans.

TRAF6 deficiency promotes TNF-induced cell death through inactivation of GSK3beta.

TNF receptor-associated factor 6 (TRAF6) plays a key role in the regulation of innate immune responses by mediating signals from both TNF receptors (TNFRs) and interleukin-1 receptors (IL-1Rs)/Toll-like receptors (TLRs). Here, we define a new role for TRAF6 in antagonizing cell death during TNF signaling. In TRAF6-deficient 3T3 (T6(-/-) 3T3) cells, TNF stimulation leads to the accumulation of reactive oxygen species (ROS), which in turn results in prolonged c-Jun N-terminal kinase (JNK) activation and accelerated cell death. Furthermore, TNF-induced p65/RelA phosphorylation as well as transcriptional activity of nuclear factor-kappaB (NF-kappaB) was significantly downregulated in T6(-/-) 3T3 cells. Interestingly, TRAF6 deficiency leads to constitutive phosphorylation and inactivation of glycogen synthase kinase 3beta (GSK3beta). Restoration of GSK3beta activity through exogenous expression of a GSK3beta constitutive active form rescued cell death in TRAF6-null 3T3 cells. These data suggest a role for TRAF6 in the maintenance of cell survival by regulating GSK3beta activity in TNF signaling.

Experimental microencapsulation of porcine and rat pancreatic islet cells with air-driven droplet generator and alginate.

Transplantation of microencapsulated islets is proposed as an ideal therapy for the treatment of type 1 diabetes mellitus without immunosuppression. This strategy is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. The aim of this study was to establish an ideal condition of microencapsulation using an air-driven droplet generator and alginate in vitro. The optimal conditions for islet encapsulation were an alginate inflow rate of 10 mL/h, CO2 flow rate of 2.0 L/min in a concentration of 2% alginate. For 2.5% alginate, the alginate inflow rate of 20 mL/h, CO2 flow rate 3.0 L/min was ideal; alginate inflow rate of 40 mL/h, CO2 flow rate of 4.0 L/min showed good microcapsules at 3% alginate. Viability of encapsulated islets was greater than 90%. In terms of insulin secretion, encapsulated islets secreted insulin in response to glucose in static culture medium. However, there was no normal response to low or high glucose challenge with a stimulation index less than 2.0. Microencapsulation of pig islets was successfully performed with air-driven droplet generator and alginate in vitro. Further studies about biocompatibility and glucose control in vivo may provide a useful tool for treatment of patients with diabetes mellitus.

Improved islet yields after purification following the novel endogenous trypsin inhibitor and histidine-tryptophan-ketoglutarate treatment in pigs.

BACKGROUND: Adult porcine islet xenotransplantation into humans is greatly diminished by the difficulty to isolate islets because of their fragility. The goal of this study was to improve the efficacy of islet yields using endogenous trypsin inhibitor and histidine-tryptophan-ketoglutarate (HTK) perfusate. METHOD: We compared two porcine islet isolation protocols: Eurocollins solution for in situ pancreas perfusion without use of an endogenous trypsin inhibitor versus HTK solution including endogenous trypsin inhibitor for pancreas perfusion. RESULTS: Endogenous trypsin inhibitor and HTK strategies significantly improved total islet yield, recovery, and islet index after purification (P < .05), whereas unpurified islet yield did not increase. An average of 228,000 +/- 95,000 islet equivalents (IEQ) (n = 20) purified islets were obtained in the first group compared with 115,000 +/- 56,000 IEQ (n = 18) in the second group. The average islet index was significantly increased in the first group compared with the second group before and after purification: before: 0.28 versus 0.49 versus after: 0.25 versus 0.4 (P < .05). At this time, islet purity, viability, and stimulation index did not show a significant difference between groups. CONCLUSION: Our study showed that endogenous trypsin inhibitor and HTK strategies significantly improved purified islet isolation efficacy because of reduction of islet fragility.

Serum neuron-specific enolase levels at presentation and long-term neurological sequelae after acute charcoal burning-induced carbon monoxide poisoning.

OBJECTIVE: This study aimed to investigate whether clinical parameters and serum neuron-specific enolase (NSE) levels measured at emergency department (ED) presentation help stratify the risk of acute or delayed persistent severe neurological sequelae after acute carbon monoxide (CO) poisoning induced by charcoal burning. METHODS: This retrospective study included 236 patients who suffered from CO poisoning. Demographic information, serum NSE levels measured in the ED, treatment, clinical course, and long-term neurological outcomes were recorded. RESULTS: The median serum NSE level at presentation was 15.5 (10.9-22.7) ng/mL. No differences were observed in the duration of CO exposure; the initial Glasgow Coma Scale (GCS) score; the levels of arterial HCO3-, white blood cells (WBCs), C-reactive protein (CRP) or troponin I; or the frequency of abnormal diffusion-weighted imaging finding at presentation among the groups with different serum NSE levels at presentation. The incidences of acute and delayed persistent neurologic sequelae assessed at 22.3 months after acute charcoal CO poisoning were 5.1% and 8.5%, respectively. No difference in the NSE level was observed between patients stratified according to long-term neurological status. According to the multinomial logistic regression analysis, age, serum CRP levels and the initial GCS score were risk factors for the two types of persistent severe neurological sequelae, whereas troponin I levels were associated only with the acute persistent severe neurological sequelae. However, the adjusted NSE level was not a risk factor for any persistent neurological sequelae. CONCLUSIONS: Serum NSE levels at presentation were not correlated with the risk of acute or delayed persistent neurological sequelae. Further studies with blood sampling at optimal time points and serial measurements should be conducted. Age, initial GCS score, and CRP levels may be risk factors for persistent severe neurological sequelae.

Laparoscopy-assisted distal gastrectomy with intracorporeal Billroth I stapled anastomosis using a hand access device for patients with gastric cancer.

BACKGROUND: Billroth I gastroduodenostomy is an anastomotic procedure used widely after gastric resection for distal gastric cancer. As laparoscopy-assisted distal gastrectomy (LADG) gains increasing popularity, various techniques of laparoscopic gastroduodenal anastomosis are being introduced. METHODS: To investigate the feasibility and benefit of their novel surgical technique of intracorporeal Billroth I stapled anastomosis using a hand access device (IBISA-HAD), the authors performed LADG using IBISA-HAD for 23 patients with distal gastric cancer and LADG using minilaparotomy Billroth I stapled anastomosis (MLBISA) for 10 patients. RESULTS: The time required for the anastomosis procedure of IBISA-HAD was 45.5 +/- 12.0 min, and the operative time, perioperative transfusion, and hospital stay were not significantly different between IBISA-HAD and MLBISA. The IBISA-HAD procedure provided a markedly enhanced vision of the stapling process, leading to less wound retraction and extension than MLBISA. CONCLUSION: The IBISA-HAD technique can provide a markedly enhanced view of the stapling procedure with the help of a current state-of-art laparoscopy system. The authors believe that this novel technique can guide an accurate laparoscopic anastomosis for the surgeon dealing with obese patients who have distal gastric cancer.

Protective effect of alpha-melanocyte-stimulating hormone on pancreas islet cell against peripheral blood mononuclear cell-mediated cytotoxicity in vitro.

The alpha-melanocyte-stimulating hormone (alpha-MSH) has been shown to interact with various cells of the immune and inflammatory systems and down-regulate either the production or the action of proinflammatory cytokines. In this study, we investigated the potential of alpha-MSH to prevent pancreatic islet cells from cytotoxic injury by inflammatory cytokines released from peripheral blood mononuclear cells (PBMCs) in rats. Pancreatic islets were cocultured with PBMCs in a transwell system during stimulation by phorbol myristic acid and ionomycin. alpha-MSH (50 nmol/L) was added to PBMCs for 2 hours before coculture. Viability and apoptosis of islets were observed by the 3-(4,5-dimethylthiazole-2-yl)-, 5-diphenyltrazolium bromide assay and flow cytometry. We measured inflammatory cytokines and nitric oxide (NO). Insulin release from islets cocultured with mononuclear cells was checked as the metric of islet function. In comparison to the control group, the viability of islets with alpha-MSH-treated mononuclear cells was increased and apoptosis reduced significantly. Inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-1beta, were significantly reduced among the alpha-MSH-treated group. NO production in the alpha-MSH-treated group was decreased significantly. Insulin secretory function of the islets recovered in conditions of alpha-MSH treatment. This study demonstrated that alpha-MSH protected pancreatic islet cells from PBMC-mediated cytotoxicity and preserved insulin secretory function. This treatment may have the potential to improve graft survival in clinical islet transplantation.

Transfection-mediated cell synchronization: acceleration of G1-S phase transition by gamma irradiation.

We have previously provided evidence that the uptake of DNA into cells is cell cycle specific following transfection. We show here that, immediately after transfection, successfully transfected cells are greatly enriched for cells in early G1 or G0 phase and that, upon removal of the DNA precipitates, cells progress through G1 and enter S phase in a synchronous fashion. We also demonstrate that this approach can be utilized in meaningful cell-cycle experiments, and we show that gamma irradiation accelerates the G1-S phase transition in a cell line with a functionally inactive p53 protein.

Anti-angiogenic activity of ursodeoxycholic acid and its derivatives.

Ursodeoxycholic acid (UDCA) and its derivatives were examined for anti-angiogenic activities by using the chick embryo chorioallantoic membrane (CAM) assay. The presence of UDCA or its derivatives inhibited angiogenesis in a dose-dependent manner; the dose required for half-maximal inhibition (ID50) was 4 microg per CAM for UDCA. The conjugate forms of UDCA with glycine methyl ester (HS-1030), L-leucine benzyl ester (HS-1068) and L-leucine methyl ester (HS-1133) were showing ID50 of 0.09, 0.08 and 0.6 microg per CAM, respectively. Also, the effect of UDCA and its derivatives on the viability of calf pulmonary artery endothelial (CPAE) cells was examined. Among them, HS-1030 effectively reduced the viability of CPAE cells in a concentration-dependent manner. Based on these results, we speculated that HS-1030 might be a promising candidate as angiogenesis inhibitor for use in the management of diseases accompanied by uncontrolled angiogenesis including solid tumor.

Circadian changes in pharmacokinetics of sulfamethoxazole administered orally to rabbits.

Circadian variations of sulfamethoxazole pharmacokinetics were studied after a single oral administration of sulfamethoxazole, 50 mg/kg, to rabbits at 09:00 (a.m.) and 22:00 (p.m.). The profiles of plasma sulfamethoxazole concentration showed from 6 h to 24 h significant statistical difference (p<0.05) between 09:00 and 22:00. The half-life (t(1/2)) was significantly shorter in the morning (11.2 +/- 3.2 h) when compared to the nighttime (15.4 +/- 3.5 h) (p< 0.05). The AUC was significantly decreased in the morning (1325 +/- 264 microg/ml x h) than that in the nighttime (2059 +/- 379 microg/ml x h) (p<0.05). Total body clearance (CLt) was significantly higher when sulfamethoxazole was given in the morning (6.65 +/- 0.23 ml/min) versus in the nighttime (4.28 +/- 0.20 ml/min) (p<0.05).

Induction of apoptosis in HepG2 human hepatocellular carcinoma cells by a novel derivative of ursodeoxycholic acid (UDCA).

The effects of ursodeoxycholic acid (UDCA) and its novel derivative, named as HS-1030, on the proliferation of HepG2, human hepatocellular carcinoma cells were investigated. Whereas UDCA had no significant effect in a concentration range we have tested, HS-1030 inhibited the proliferation of HepG2 cells in a concentration dependent manner. Surprisingly, HS-1030 had no effect on the proliferation of Human Chang liver cell which is a normal liver cell line. We also found that proliferation-inhibitory effect of HS-1030 was due to the induction of apoptosis of HepG2 cells, which was confirmed by observing the internucleosomal DNA fragmentation and morphological changes (i.e. cell shrinkage, nuclear condensation and the formation of apoptotic bodies). These results suggest that HS-1030 may be a good candidate as a drug for the treatment of liver cancer.

Alpha-melanocyte stimulating hormone preserves islet graft survival through down-regulation of Toll-like receptors.

The induction of Toll-like receptors (TLRs) in ß cells is involved in ß-cell death and graft rejection after transplantation. This study investigated the ability of alpha-melanocyte stimulating hormone (α-MSH) to protect pancreatic islets and improve graft survival through regulation of TLRs. To test the effect of α-MSH on TLR regulation, we first isolated pancreatic islets from rats pretreated with/without α-MSH and assayed inflammatory cytokines and insulin release, and measured the expression of TLRs. Pancreatic islets were transplanted into the kidney capsule of a diabetes mellitus (DM) mouse with and without prior injection of α-MSH. The blood glucose levels were measured and TLR4 expression in transplanted kidney tissue was assessed. Islet morphology, including size and total mass, was improved in the α-MSH group compared to the control group. The expression of TLRs as well as nitric oxide and monocyte chemoattractant protein 1 production were decreased in islets isolated from α-MSH-treated rats. In DM mice, the normoglycemic ratio was higher in the α-MSH-treated group than in the sham group. Moreover, the high levels of TLR4 expression observed in DM kidney tissue were significantly decreased in islet-transplanted tissue with α-MSH. This study showed that α-MSH protects pancreatic islets from cell death and dysfunction through downregulation of TLRs. In conclusion, α-MSH could contribute to improved islet graft survival and function in pancreatic islet transplantation.

Cell cycle analysis of Epstein-Barr virus-infected cells following treatment with lytic cycle-inducing agents.

While Epstein-Barr virus (EBV) latency-associated gene expression is associated with cell cycle progression, the relationship between the EBV lytic program and the cell cycle is less clear. Using four different EBV lytic induction systems, we address the relationship between lytic cycle activation and the cell cycle. In three of these systems, G0 or G1 cell growth arrest signaling is observed prior to detection of the EBV immediate-early gene product Zta. In tetradecanoyl phorbol acetate-treated P3HR1 cultures and in 5-iodo-2'-deoxyuridine-treated NPC-KT cultures, cell cycle analysis of Zta-expressing cell populations showed a significant G1 bias during the early stages of lytic cycle progression. In contrast, treatment of the cell line Akata with anti-immunoglobulin (Ig) results in rapid induction of immediate-early gene expression, and accordingly, activation of the immediate-early gene product Zta precedes significant anti-Ig-induced cell cycle effects. Nevertheless, cell cycle analysis of the Zta-expressing population following anti-Ig treatment shows a bias for cells in G1, indicating that anti-Ig-mediated induction of Zta occurs more efficiently in cells traversing G1. Last, although 5-azacytidine treatment of Rael cells results in a G1 arrest in the total cell population which precedes the induction of Zta, cell cycle analysis of the Zta-expressing population shows a significant bias for cells with an apparent G2/M DNA content. This bias may result, in part, from activation of Zta expression following demethylation of the Zta promoter during S-phase. Together, these studies indicate that induction of Zta occurs through several distinct mechanisms, some of which may involve checkpoint signaling.

Multiple phosphorylation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B by casein kinase II and p60c-src in vitro.

We have cloned a soluble chicken protein tyrosine phosphatase, named CPTP1, from the cDNA library of chicken intestine. The CPTP1 showed 92% sequence identity to the corresponding 321 amino acid residues of human PTP1B (HPTP1B). CPTP1 lacked 13 amino acids of the N-terminal region compared with HPTP1B, while the C-terminal 48 amino acid sequence of this protein was distinct from those of other PTPs. In vitro phosphorylation and phosphoamino acid analysis showed that both CPTP1 and HPTP1B were phosphorylated on serine and threonine residues near their N-terminus by casein kinase II (CKII). Furthermore, phosphorylation of CPTP1 by CKII resulted in an inhibition of its phosphatase activity in vitro. Interestingly, both CPTP1 and HPTP1B were also tyrosine-phosphorylated near their N-terminus by p60c-src. When we examined the vanadate effect, in the absence of vanadate, the tyrosine-phosphorylated CPTP1 by p60c-src was autodephosphorylated by its own phosphatase activity. These results suggest that both CPTP1 and HPTP1B might play an important role in CKII- and p60c-src-induced signal transduction cascades.