Sensitivity of the Wound Edge Gene Signature "WD14" in Responding to Clinical Change: A Longitudinal Cohort Study.

Introduction: WounD14 (WD14) gene signature is a recently developed tool derived from genetic interrogation of wound edge biopsies of chronic venous leg ulcers to identify heard-to-heal wounds and enable clinicians to target aggressive therapies to promote wound healing. This study aimed to evaluate if changes in wound clinical healing status were detected by the WD14 gene signature over time as this is currently poorly understood. Material and methods: WD14 was developed through gene screening and subsequent validation in 3 patient cohorts involving 85 consecutive patients with chronic venous leg ulcers referred to a tertiary wound healing unit. Patients underwent a wound edge biopsy to interrogate for a "healing" or "non-healing" genotype. A smaller cohort (18%) underwent a second biopsy, which comprised this pilot cohort reported herein. Twelve weeks following biopsy, wounds were clinically assessed for healing status based on reduction in size and compared to WD14 genotype. Results: Sequential biopsies and WD14 scores were derived from 16 patients. WD14 signature predicted wound healing status among this cohort at either visit (32 wound edge biopsies) with a positive predictive value (PPV) of 85.2% (95% CI 74.1%-92.0%) and negative predictive value (NPV) of 80.0% (95% CI 34.2%-96.9%). A total of 6 wounds underwent altered clinical status between the 2 visits. In this cohort, WD14 has a PPV of 66.7% (95% CI 47.3%-81.7%) and NPV of 100%. Conclusion: Although the WD14 gene signature did change with wound healing status, larger studies are required to precisely clarify its role and ability to prognosticate wounds of differing clinical status over time.

Quantitative analysis of gene expression changes in response to genotoxic compounds.

Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.

Earthworms produce phytochelatins in response to arsenic.

Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species) as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

Induction of expression of a 14-3-3 gene in response to copper exposure in the marine alga, Fucus vesiculosus.

The macro-alga Fucus vesiculosus has a broad global and estuarine distribution and exhibits exceptional resistance to toxic metals, the molecular basis of which is poorly understood. To address this issue a cDNA library was constructed from an environmental isolate of F. vesiculosus growing in an area with chronic copper pollution. Characterisation of this library led to the identification of a cDNA encoding a protein known to be synthesised in response to toxicity, a full length 14-3-3 exhibiting a 71% identity to human/mouse epsilon isoform, 70-71% identity to yeast BMH1/2 and 95 and 71% identity to the Ectocarpus siliculosus 14-3-3 isoforms 1 and 2 respectively. Preliminary characterisation of the expression profile of the 14-3-3 indicated concentration- and time-dependent inductions on acute exposure of F. vesiculosus of copper (3-30 µg/l). Higher concentrations of copper (≥150 µg/l) did not elicit significant induction of the 14-3-3 gene compared with the control even though levels of both intracellular copper and the expression of a cytosolic metal chaperone, metallothionein, continued to rise. Analysis of gene expression within environmental isolates demonstrated up-regulation of the 14-3-3 gene associated with the known copper pollution gradient. Here we report for the first time, identification of a gene encoding a putative 14-3-3 protein in a multicellular alga and provide preliminary evidence to link the induction of this 14-3-3 gene to copper exposure in this alga. Interestingly, the threshold exposure profile may be associated with a decrease in the organism's ability to control copper influx so that it perceives copper as a toxic response.

Determination of gene expression patterns using in situ hybridization to Drosophila testes.

We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult flies, fixed and processed for hybridization. Both probe and fixed testes can be stored before use. Extensive post-hybridization washing reduces the background. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction. This protocol is suitable for low-medium throughput applications with parallel processing of 2-48 samples, and takes 4-5 d to complete. We have used this protocol, which is similar to other RNA ISH protocols, but optimized for whole-mount Drosophila testes, to document the expression of about 1,000 genes in Drosophila melanogaster male genital tract.

Comparison of transcription-mediated amplification and growth-based methods for the quantitation of Enterococcus bacteria in environmental waters.

An assay based on transcription-mediated amplification (TMA) technology was used to quantitate Enterococcus fecal indicator bacteria in environmental water samples. The results generated by this and two growth-based methods relative to the 104 most-probable-number or CFU-per-100-ml threshold show that the three methods are in good qualitative agreement when tested against a range of water samples taken from different locations. The results demonstrate sensitive and rapid detection (approximately 4 h from sample collection to result) and quantitation of Enterococcus bacteria compared to the results with the growth-based methods.