Toehold-mediated internal control to probe the near-field interaction between the metallic nanoparticle and the fluorophore.

Metallic nanoparticles (MNPs) are known to alter the emission of vicinal fluorophores through the near-field interaction, leading to either fluorescence quenching or enhancement. Much ambiguity remains in the experimental outcome of such a near-field interaction, particularly for bulk colloidal solution. It is hypothesized that the strong far-field interference from the inner filter effect of the MNPs could mask the true near-field MNP-fluorophore interaction significantly. Thus, in this work, a reliable internal control capable of decoupling the near-field interaction from far-field interference is established by the use of the DNA toehold concept to mediate the in situ assembly and disassembly of the MNP-fluorophore conjugate. A model gold nanoparticle (AuNP)-Cy3 system is used to investigate our proposed toehold-mediated internal control system. The maximum fluorescence enhancement is obtained for large-sized AuNP (58 nm) separated from Cy3 at an intermediate distance of 6.8 nm, while fluorescence quenching is observed for smaller-sized AuNP (11 nm and 23 nm), which is in agreement with the theoretical values reported in the literature. This work shows that the toehold-mediated internal control design can serve as a central system for evaluating the near-field interaction of other MNP-fluorophore combinations and facilitate the rational design of specific MNP-fluorophore systems for various applications.

The FOXM1-PLK1 axis is commonly upregulated in oesophageal adenocarcinoma.

BACKGROUND: The transcription factor FOXM1 is an important regulator of the cell cycle through controlling periodic gene expression during the G2 and M phases. One key target for FOXM1 is the gene encoding the protein kinase PLK1 and PLK1 itself acts in a positive feedback loop to phosphorylate and activate FOXM1. Both FOXM1 and PLK1 have been shown to be overexpressed in a variety of different tumour types. METHODS: We have used a combination of RT-PCR, western blotting, tissue microarrays and metadata analysis of microarray data to study whether the FOXM1-PLK1 regulatory axis is upregulated and operational in oesophageal adenocarcinoma. RESULTS: FOXM1 and PLK1 are expressed in oesophageal adenocarcinoma-derived cell lines and demonstrate cross-regulatory interactions. Importantly, we also demonstrate the concomitant overexpression of FOXM1 and PLK1 in a large proportion of oesophageal adenocarcinoma samples. This co-association was extended to the additional FOXM1 target genes CCNB1, AURKB and CKS1. In a cohort of patients who subsequently underwent surgery, the expression of several FOXM1 target genes was prognostic for overall survival. CONCLUSIONS: FOXM1 and its target gene PLK1 are commonly overexpressed in oesophageal adenocarcinomas and this association can be extended to other FOXM1 target genes, providing potentially important biomarkers for predicting post-surgery disease survival.

PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma.

BACKGROUND: Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells. METHODS: Immunohistochemical patterns of PEA3 expression and active ERK signalling were analysed and mRNA expression levels of PEA3, ER81, MMP-1 and MMP-7 were determined in gastric adenocarcinoma samples. RESULTS: Here, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in gastric adenocarcinomas. PEA3 is upregulated at the protein level in gastric adenocarcinomas and both PEA3/ETV4 and ER81/ETV1 are upregulated at the mRNA level in gastric adenocarcinoma tissues. This increased expression correlates with the expression of a target gene associated with metastasis, MMP-1. Enhanced ERK signalling is also more prevalent in late-stage gastric adenocarcinomas, and the co-association of ERK signalling and PEA3 expression also occurs in late-stage gastric adenocarcinomas. Furthermore, the co-association of ERK signalling and PEA3 expression correlates with decreased survival rates. CONCLUSIONS: This study shows that members of the PEA3 subfamily of transcription factors are upregulated in gastric adenocarcinomas and that the simultaneous upregulation of PEA3 expression and ERK pathway signalling is indicative of late-stage disease and a poor survival prognosis.

EndoCinch treatment for gastro-oesophageal reflux disease.

Gastro-oesophageal reflux disease (GORD) is a common disorder which significantly impairs the quality of life. Recently an EndoCinch system has been described, with an approach to the treatment of GORD that would obviate the need for long-term proton pump inhibitors and the cost and potential risk of laparoscopic Nissen fundoplication. We set outto evaluate the status of this new technique for the management of GORD. We review the literatures (publications and abstracts) regarding safety, efficacy, and durability of this new antireflux procedure. On the whole, this new antireflux technique produced significant improvement in GORD symptomatology and quality of life and reduced the use of antireflux medication. However, it failed to normalize acid reflux, long-term durability data are lacking, and some serious side effects have been reported. In conclusion, EndoCinch has the potential to treat patients with this common ailment. However, further studies are necessary to determine what modifications to this antireflux technique are required in order to produce the maximum clinical benefit.

Human peripheral and gastric lymphocyte responses to Helicobacter pylori NapA and AphC differ in infected and uninfected individuals.

BACKGROUND: In this study, we identify the nature of the immunological response of human peripheral blood mononuclear cells (PBMC) and lamina propria gastric lymphocytes (LPL) to two Helicobacter pylori antigens, the neutrophil activating protein (NapA) and alkyl hydroperoxide reductase (AphC). These antigens were identified and selected for study based on the observation that serological recognition of these proteins was associated with H pylori negative status in humans. AIMS: The aim was to study the serological, proliferative, and cytokine responses of PBMC and LPL, obtained from H pylori infected and uninfected individuals, to these antigens. METHODS: Patient serum, PBMC, and LPL were used to determine antibody isotype, and proliferative and cytokine responses to recombinant forms of NapA and AphC using western blotting and ELISA. RESULTS: Western blotting revealed antibody reactivity to recombinant NapA and AphC among the H pylori negative population studied. Both the proliferative and interferon gamma responses of PBMC and LPL to NapA and AphC were significantly higher in H pylori negative compared with H pylori positive subjects. Analysis of the IgG subclass profiles to both antigens revealed a T helper 1 associated IgG3 antibody response in uninfected individuals. However, interleukin 10 production was greater in H pylori positive individuals in response to these antigens. CONCLUSIONS: Taken together these data are consistent with an immune response to these antigens skewed towards a T helper 1 response in the uninfected cohort.

Helicobacter pylori activates the early growth response 1 protein in gastric epithelial cells.

The early growth response 1 (Egr-1) transcription factor is rapidly induced by various stimuli and is implicated in the regulation of cell growth, differentiation, and gene expression. The aim of this study was to examine the effect of Helicobacter pylori on the expression of Egr-1 and Egr-1-regulated genes in gastric epithelial AGS cells. Egr-1 expression was assayed by immunoblotting and electrophoretic mobility shift assays using H. pylori-stimulated AGS cells. Transient transfection experiments with promoter-reporter constructs of CD44, ICAM-1, and CD95L were used for expression studies. H. pylori induced the expression of Egr-1 in gastric epithelial cell lines in a dose-dependent manner, with the rapid kinetics that are typical of this class of transcription factors. Immunohistochemical studies of biopsies revealed that Egr-1 expression is more abundant in H. pylori-positive patients than in uninfected individuals. Reporter-promoter transfection studies indicated that Egr-1 binding is required for the H. pylori-induced transcriptional promoter activity of the CD44, ICAM-1, and CD95L (APO-1/Fas) constructs. The blocking of egr-1 with an antisense sequence prevented H. pylori-induced Egr-1 and CD44 protein expression. The MEK1/2 signaling cascade participates in H. pylori-mediated Egr-1 expression, but the p38 pathway does not. The data indicate that H. pylori induces Egr-1 expression in AGS cells in vitro and that the Egr-1 protein is readily detectable in biopsies from H. pylori-positive subjects. These observations suggest that H. pylori-associated Egr-1 expression may play a role, in part, in H. pylori-induced pathology.

Randomized comparison of unfractionated heparin with corticosteroids in severe active inflammatory bowel disease.

BACKGROUND: Heparin therapy may be effective in steroid resistant inflammatory bowel disease. AIM: A randomized pilot study, to compare unfractionated heparin as a first-line therapy with corticosteroids in colonic inflammatory bowel disease. METHODS: Twenty patients with severe inflammatory bowel disease (ulcerative colitis, n=17; Crohn's colitis, n=3) were randomized to either intravenous heparin for 5 days, followed by subcutaneous heparin for 5 weeks (n=8), or high-dose intravenous hydrocortisone for 5 days followed by oral prednisolone 40 mg daily, reducing by 5 mg per day each week (n=12). After 5 days, non-responders in each treatment group were commenced on combination therapy. Response to therapy was monitored by: clinical disease activity (ulcerative colitis: Truelove and Witt Index; Crohn's colitis: Harvey and Bradshaw Index), stool frequency, serum C-reactive protein and alpha1 acid glycoprotein, endoscopic and histopathological grading. RESULTS: The response rates were similar in both treatment groups: clinical activity index (heparin vs. steroid; 75% vs. 67%; P=0.23), stool frequency (75% vs. 67%; P=0.61), endoscopic (75% vs. 67%; P=0.4) and histopathological grading (63% vs. 50%; P=0.67). Both treatments were well-tolerated with no serious adverse events. CONCLUSION: Heparin as a first line therapy is as effective as corticosteroids in the treatment of colonic inflammatory bowel disease. Large multicentre randomized comparative studies are required to determine the role of heparin in the management of inflammatory bowel disease.