The prognostic and therapeutic value of EpHA2 in early colorectal cancer (CRC).

Background: EpHA2 is a 130 kD transmembrane glycoprotein belonging to ephrin receptor subfamily and involved in angiogenesis/tumour neovascularisation. High EpHA2 mRNA level has recently been implicated in cetuximab resistance. Previously, we found high EpHA2 levels in a panel of invasive colorectal cancer (CRC) cells, which was associated with high levels of stem-cell marker CD44. Our aim was to investigate the prognostic value of EpHA2 and subsequently correlate expression levels to known clinico-pathological variables in early stage CRC. Methods: Tissue samples from 509 CRC patients were analysed. EpHA2 expression was measured using IHC. Kaplan-Meier graphs were used. Univariate and multivariate analyses employed Cox Proportional Hazards Ratio (HR) method. A backward selection method (Akaike’s information criterion) was used to determine a refined multivariate model. Results: EpHA2 was highly expressed in CRC adenocarcinoma compared to matched normal colon tissue. In support of our preclinical invasive models, strong correlation was found between EpHA2 expression and CD44 and Lgr5 staining (p<0.001). In addition, high EpHA2 expression significantly correlated with vascular invasion (p=0.03).HR for OS for stage II/III patients with high EpHA2 expression was 1.69 (95%CI: 1.164-2.439; p=0.003). When stage II/III was broken down into individual stages, there was significant correlation between high EpHA2 expression and poor 5-years OS in stage II patients (HR: 2.18; 95%CI: 1.28-3.71; p=0.005).HR in the stage III group showed a trend to statistical significance (HR: 1.48; 95%CI=0.87-2.51; p=0.05). In both univariate and multivariate analyses of stage II patients, high EpHA2 expression was the only significant factor and was retained in the final multivariate model. Higher levels of EpHA2 were noted in our RAS and BRAF mutant CRC cells, and silencing EpHA2 resulted in significant decreases in migration/invasion in parental and invasive CRC sublines. Correlation between KRAS/NRAS/BRAFmutational status and EpHA2 expression in clinical samples is ongoing. Conclusions: Taken together, our study is the first to indicate that EpHA2 expression is a predictor of poor clinical outcome and a potential novel target in early stage CRC.

MMP-8 Activity Profiling In GCF Samples

Background: In healthy tissues a family of enzymes known as matrix metalloproteinases (MMPs) play an important role in regulating turnover and metabolism of connective tissue collagen. MMPs have been implicated in a wide variety of pathological conditions including periodontal disease. MMP-8 has been extensively studied in periodontal health and disease using enzyme-linked immunosorbent assay (ELISA). Although ELISA quantifies the presence of the MMP-8 protein, it is not possible to determine enzyme activity using this method. Furthermore, since members of the MMP family have poor substrate sequence specificity, a peptide substrate alone cannot differentiate the activity of MMP-8 from other MMPs that may be present in biological samples. Objectives: In the present study, a method to specifically measure MMP-8 activity in gingival crevicular fluid (GCF) samples was developed. Methods: GCF was collected from healthy patients and those with periodontal disease using Perio paper strips. Samples were stored frozen until required for analysis. A specific MMP-8 antibody was used to coat 96 well microtitre plates to selectively remove MMP-8 from the GCF samples. Following a washing step, the activity of bound MMP-8 was measured over 70 minutes using a fluorogenic (FRET) substrate. Results: GCF from healthy subjects exhibited basal MMP-8 activity but in diseased samples MMP-8 activity was significantly higher. Minimal binding of other recombinant MMPs to the specific MMP-8 antibody was observed in cross-reactivity studies. Conclusion: We show for the first time that MMP-8 activity was significantly increased in GCF from periodontitis sites compared with activity levels in healthy sites. Further studies of MMP-8 activity in GCF samples should improve our understanding of its destructive role in periodontal disease.

Identification of human neutrophil defensins (HNPs1-3) in biopsies of squamous cell carcinomas of the human tongue

It has previously been reported that the a-defensins, found in the granules of polymorphonuclear leukocytes (neutrophils/ PMNs), are cytolytic for human tumour cells in vitro. Objective: To identify and quantify the a- defensins, HNP-1, HNP-2 and HNP-3 in healthy and tumour tissue from patients with oral squamous cell carcinoma using HPLC, mass spectrometry and amino acid sequencing. Methods: All patients (n=5) were diagnosed with oral squamous cell carcinoma of the tongue.Biopsy tissue from the site of the tumour (n=5) and a non-affected region of the tongue (n=5) was snap frozen and subsequently stored at -70 ºC until analysed. Peptides were extracted from the 10 tissue biopsies using acidified ethanol. Peptide extracts were separated by reverse-phase HPLC . All tumour and control tissue samples were individually analysed under identical conditions with a flow rate of l ml/min, ambient column temperature and absorbance detection at 214 and 280 nm. Fractions (1ml) were collected automatically. HPLC fractions were analysed by MALDI-MS using a linear time-of-flight Voyager DE-mass spectrometer (PerSeptive Biosystems, UK). Using this system the detection limit was 10 fmol. Peptides with molecular masses corresponding to those reported for the a-defensins were deemed of interest and were further subject to complete structural analysis by automated Edman degradation using an Applied Biosystems 491 Procise microsequencer. Results: MALDI-MS revealed a triad of peptides of molecular masses 3442 Da, 3371 Da and 3486 Da in both healthy and tumour tissue. Full length sequence data were obtained for the three a-defensins, unequivocally identifying their presence in both tumour and healthy tissue. Analysis of the MALDI-MS and sequence data indicated that the a-defensins were overexpressed (up to 12 fold) in tumour tissue. Conclusion: This study demonstrates the feasibility of screening tumour tissue for novel peptides/proteins using HPLC and MALDI-MS.The role of a-defensins in oral squamous cell carcinoma of the tongue requires further investigation.

Geographical variation in inorganic arsenic in paddy field samples and commercial rice from the Iberian Peninsula

This study investigated total arsenic and arsenic speciation in rice using ion chromatography with mass spectrometric detection (IC-ICP-MS), covering the main rice-growing regions of the Iberian Peninsula in Europe. The main arsenic species found were inorganic and dimethylarsinic acid. Samples surveyed were soil, shoots and field-collected rice grain. From this information soil to plant arsenic transfer was investigated plus the distribution of arsenic in rice across the geographical regions of Spain and Portugal. Commercial polished rice was also obtained from each region and tested for arsenic speciation, showing a positive correlation with field-obtained rice grain. Commercial polished rice had the lowest i-As content in Andalucia, Murcia and Valencia while Extremadura had the highest concentrations. About 26% of commercial rice samples exceeded the permissible concentration
for infant food production as governed by the European Commission. Some cadmium data is also presented, available with ICP-MS analyses, and show low concentration in rice samples.

Infrared and Raman screening of seized novel psychoactive substances: A large scale study of >200 samples

The potential of IR absorption and Raman spectroscopy for rapid identification of novel psychoactive substances (NPS) has been tested using a set of 221 unsorted seized samples suspected of containing NPS. Both IR and Raman spectra showed large variation between the different sub-classifications of NPS and smaller, but still distinguishable, differences between closely related compounds within the same class. In initial tests, screening the samples using spectral searching against a limited reference library allowed only 41% of the samples to be fully identified. The limiting factor in the identification was the large number of active compounds in the seized samples for which no reference vibrational data were available in the libraries rather than poor spectral quality. Therefore, when 33 of these compounds were independently identified by NMR and mass spectrometry and their spectra used to extend the libraries, the percentage of samples identified by IR and Raman screening alone increased to 76%, with only 7% of samples having no identifiable constituents. This study, which is the largest of its type ever carried out, therefore demonstrates that this approach of detecting non-matching samples and then identifying them using standard analytical methods has considerable potential in NPS screening since it allows rapid identification of the constituents of the majority of street quality samples. Only one complete feedback cycle was carried out in this study but there is clearly the potential to carry out continuous identification/updating when this system is used in operational settings.

 Graphitization of Small Carbonate Samples for Paleoceanographic Research at the Godwin Radiocarbon Laboratory, University of Cambridge

A new radiocarbon preparation facility was set up in 2010 at the Godwin Laboratory for Palaeoclimate Research, at the University of Cambridge. Samples are graphitized via hydrogen reduction on an iron powder catalyst before being sent to the Chrono Centre, Belfast, or the Australian National University for accelerator mass spectrometry (AMS) analysis. The experimental setup and procedure have recently been developed to investigate the potential for running small samples of foraminiferal carbonate. By analyzing background values of samples ranging from 0.04 to 0.6 mg C along with similar sized secondary standards, the setup and experimental procedures were optimized for small samples. “Background” modern 14C contamination has been minimized through careful selection of iron powder, and graphitization has been optimized through the use of “small volume” reactors, allowing samples containing as little as 0.08 mg C to be graphitized and accurately dated. Graphitization efficiency/fractionation is found not to be the main limitation on the analysis of samples smaller than 0.07 mg C, which rather depends primarily on AMS ion beam optics, suggesting further improvements in small sample analysis might yet be achieved with our methodology.